Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic airway infections, a major determinant of lung inflammation and damage in cystic fibrosis (CF) patients. P.aeruginosa employs chemical signals to communicate between cells in a system called quorum sensing(QS), using several hierarchical and arranged interconnected QS circuits to regulate the expression of many genes, including those that code for virulence factors. I investigated two interconnected QS systems in P.aeruginosa, the LasR-LasI system and RhlR-RhlI system, to determine the QS hierarchy in P.aeruginosa. In the lab strain of P.aeruginosa, PAO1, the QS transcription factor LasR sits atop the hierarchy and activates transcription for many genes, including the one that encodes the QS regulator RhlR. This arrangement leads to a “hierarchy” where genes regulated by RhlR also require LasR activity. However, CF isolates have an alternate system where RhlR acts in a LasR-independent manner. We formulated the hypothesis that there may in fact be multiple architectures of quorum sensing in P.aeruginosa. The primary inquiry of this study revolves around whether clinical isolates of P.aeruginosa obtained from chronic CF infections would demonstrate continued activity of RhlR when lasR is deleted. To delete lasR, I used an allelic-exchange approach and made LasR mutants in 6 CF strains. By knocking out the lasR gene and making GFP reporter strains via electroporation, we measure RhlR and LasR activities. We found that the hierarchy as described in PAO1 is not a feature of all strains. Also, using whole-genome sequencing, we found that genes implicated in generating the hierarchy are not required in clinical strains. The findings of this project allow us to fully understand the QS hierarchy of P. aeruginosa in patients with CF, which is to determine targets for therapeutic development to combat P. aeruginosa present in patients.

I am interested in producing Human Papillomavirus (HPV) type specific monoclonal antibodies (mAbs) from B cells derived from adolescent females and young women vaccinated with the licensed human papillomavirus vaccine, Gardasil 9, which provides protection against HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58. Fluorescently labeled pseudoviruses for these nine HPV types, as well as phenotypic markers of memory B cells are used to isolate HPV specific memory B cells via fluorescence-activated cell sorting (FACs). Gene transcripts for Immunoglobulin (Ig) heavy and corresponding light chain variable regions are amplified through Reverse Transcription Polymerase Chain Reaction (RT-PCR), cloned into their respective IgG1 backbone vectors, and subsequently transfected into eukaryotic cells to produce antibodies. HPV types [6, 11, 16, and 18] have been previously produced by our lab. I aim to expand on this work by developing antibodies against types 31, 33, 45, 52, and 58. I will characterize these mAbs for HPV binding and neutralization. Production of these well-characterized, type specific antibodies are useful for HPV studies, because they provide standards in HPV serologic assays. 

Family businesses are some of the most important economic contributors in the United States, accounting for approximately 64% of the U.S. GDP. The family business model, which refers to any business with two or more family members on the board or in ownership, is a crucial and enduring part of business in the Seattle area and abroad. Historians have often pointed out that the family business model seems to be the base model for business and has thus been present since the beginning of organized business, often in the form of farms, merchant companies, banks, and other small businesses. Despite its prevalence, the family business model is far from perfect because of its numerous commonly encountered limitations. One of the limitations family businesses face is the challenge of succession, as only about 30% are able to succeed from the first generation to the second. Other limitations relate to growth, sustainability, and qualification problems. This study, conducted as a literature review, uses a combination of peer-reviewed articles and popular sources (chosen based on criteria of relevancy and prominence) as quantitative data to examine the consensus of family businesses in Seattle and the solutions that have been proposed to address these limitations. Interviews with family business owners in the Seattle area were also conducted to provide qualitative data and to highlight specific opinions. The economic and historical implications of Seattle family business are also discussed. This research aims to provide insight into otherwise costly financial, succession, and leadership difficulties in order to ensure that the family business model is an enduring contributor to the Seattle economy. Having the proper knowledge on how to approach these difficulties and reconcile with their seemingly conflicting nature can help family businesses in the Seattle area thrive while working through complicated business situations.

An important part of addiction recovery is degrading high value associations between drug cues and the drugs themselves. Dopamine plays a crucial role in learning, and is specifically implicated in the prefrontal cortex (PFC) and reversal learning - learning to update and change behavior when it is no longer being rewarded. Past studies have reported elevations in dopamine during contingency reversal, but the timescale of how activity of PFC dopamine neurons maps to reversal learning remains unclear. Here we investigated the activity of PFC dopamine during reversal learning in a longitudinal fiber photometry study, recording dopamine signal on a timescale of seconds. Mice were trained on a reversal learning task where they initially learned that two of four presented odors precipitated a sucrose reward in 85% of the trials while the remaining two odors precipitated the reward for only 15% of the trials. Once the learning was stable, reward probability flipped for two odors (one 85% odor and one 15% odor) and the mice had to update their behavior to the new odor/reward structure. Fiber photometry recordings were conducted during pre-reversal, reversal, and post-reversal stages of the study. Our data replicate findings demonstrating elevated dopamine release during the reversal period, centered around the 15-85 cue. Analysis of the relationship between the dopamine signal and behavior also revealed significant cue, reward prediction error, and 15-85 reversal coding in the majority of animals, suggesting a multi-faceted role for dopamine in the PFC. Given this, dopamine in the PFC may play an important mediating role in the enhancement of associations between drugs and drug cues, but does not play a clear role in contingency degradation.

Two-dimensional (2D) materials are layered materials made up of either a single or very few atomic layers. The properties they exhibit are fundamentally different from those of three-dimensional crystals, and they provide us with high levels of control to design novel systems in order to study new physical phenomena. 2D dielectrics are used in electrostatic gating to control the displacement field and carrier doping being applied to a 2D sample, making the properties and performance of 2D dielectrics very important. In 2D heterostructure devices, the standard dielectric used is hexagonal boron nitride (hBN). However, a recently published paper  claims that the novel 2D dielectric Bi₂SeO₅ is readily exfoliated, transferrable, and has a dielectric constant of 15, significantly higher than that of hBN, which ranges between 3-4. In this project I aimed to determine the dielectric constant and breakdown characteristics of Bi₂SeO₅ in hopes of finding a more effective substitute for hBN. I successfully measured the dielectric constant of Bi₂SeO₅ by fabricating a 2D graphene heterostrcuture device and taking graphene transport measurements comparing hBN and Bi₂SeO₅. From the data obtained I found the dielectric constant to be 14.3, which agrees with literary values. I fabricated a Bi₂SeO₅ backgate using a dry transfer technique in order to perform characterize the breakdown characteristics of the dielectric. By applying a voltage to each contact, grounding the gates, and then floating all other contacts, I measured the current passing through the dielectric until we began to see an exponential trend in order to measure breakdown. This is of interest because if Bi₂SeO₅ is proven to be more effective than hBN, it would let us apply a higher displacement field and doping to samples which would let us access new, exotic phases in 2D materials such as WTe₂.

As our world progresses through technological advancements, much of our planet regresses as an effect of climate change, highlighting a need for underutilized resources to be brought to the forefront of industry. One avenue for transforming abundant resources into useful chemicals for generating sources like fuel is the catalytic upgrading of biomass derived molecules. However, catalysts traditionally used for these reactions are not stable to contaminants in biomass mixtures, such as water or organic acids. For biomass derived molecules to serve as precursors for biofuel and other related energy sources, more stable and efficient catalysts are needed. Our group has recently shown that a bifunctional acid–base MOF with co-localized acidic and basic sites outperforms a MOF with randomly dispersed acid–base sites for the aldol condensation reaction. To further demonstrate the importance of having the acid and base groups co-localized, I synthesized and tested three control frameworks for comparison: (1) a framework with no functionality, (2) a framework with only acidic sites, and (3) a framework with only basic sites. I then tested stability and recyclability of the bifunctional acid–base frameworks by conducting recycling experiments. I resubjected the same sample to reaction conditions for a total of 5 cycles. After each cycle, I used 1H NMR to quantify the conversion of starting material to ensure that there were no changes in catalytic activity. Lastly, I used powder X-ray diffraction (PXRD) to ensure that the catalysts maintained their crystalline structure after 5 cycles. Here I show that metal–organic frameworks (MOFs), a class of porous crystalline solids, can be used as efficient and recyclable catalysts for the aldol condensation, an important reaction for biomass conversion. Overall, this work illustrates the stability and reusability of metal organic frameworks as catalysts and thus their potential for utility in biomass upgrading reactions.

The sensation of acute pain is fundamental to survival, indicating tissue damage that motivates an animal to engage in adaptive protective behaviors. Chronic pain, however, is pain that persists beyond the typical recovery window and serves little adaptive function. Due to the negative affective component inherent in chronic pain, it has been shown to contribute to the development of comorbid psychiatric disorders such as depression, social aggression, and social withdrawal. Our research aims to understand how acute and chronic pain affects social reward, motivation, and behavior. Our experiments used social self-administration (SSA), where pair-housed mice were placed in operant chambers and underwent voluntary lever press trials for the reward of social interaction with their cagemate. Following the acquisition of the SSA task, male and female mice then received a spared nerve injury (SNI) to induce neuropathic pain. Following recovery from the SNI surgery, experimental mice were returned to the lever press trials at different post-operative windows to simulate acute and persistent pain windows. Changes in social behavior were measured via changes in lever press frequency and interactive behavior during trials. Our data shows that male and female mice who underwent SSA trials immediately following nerve ligation maintained normal levels of social interaction. In contrast, female experimental animals that began SSA at a delayed time point, simulating persistent pain, lever pressed significantly less for a social partner compared to their respective control group. Conversely, male experimental animals at the delayed time point displayed high lever pressing behavior for a social partner. This data points towards divergent social adaptations following pain due to the sex of the experimental mice. Future experiments will further delineate these sex-specific adaptations following a traumatic injury. This research can inform social intervention as an adjunct or alternative treatment to pharmacological pain intervention and its comorbidities.

Mitochondria are the main oxygen consumers in eukaryotic cells and as such are the primary organelles affected by oxygen deprivation, hypoxia. Hypoxia alters the size and shape of mitochondria, called the mitochondrial dynamics, but their role in hypoxic cell death is unknown. The Crowder lab has recently discovered that a mutation in the Mechanistic Target of Rapamycin Complex One (mTORC1) protein Raptor confers hypoxia resistance in the nematode C. elegans. mTORC1 is a master regulator of metabolism and is known to affect certain aspects of mitochondrial biology. Given these two facts, we tested the hypothesis that the hypoxia resistance of the C. elegans Raptor mutant is from alterations of mitochondrial dynamics. First, I showed that hypoxia induces small, rounded mitochondria in C. elegans caused from mitochondrial fission. Second consistent with the hypothesis, I showed that the mitochondria appear to have more normal morphology before and after hypoxia in the Raptor mutant. However, not consistent with the hypothesis, a C. elegans mutant with excess mitochondrial fission was not hypersensitive to hypoxia. Then combining the hyper fission mutant with the Raptor mutant did not diminish the hypoxia resistance produced by reduced Raptor function. Thus, our data demonstrates abrogating mitochondrial fission is not necessary for the hypoxia resistance produced by the Raptor mutant and leads us to reject our hypothesis. By exploring the interaction of mitochondrial fusion and fission with Raptor, we are beginning to understand how these important organelle and metabolic regulators combine to control hypoxic cell death.

This study explores the adaptive dynamics of Pseudomonas aeruginosa (P. aeruginosa), a bacterium that often plagues cystic fibrosis patients. In P. aeruginosa, many genes are regulated through quorum sensing (QS), a form of cell-to-cell communication. In wild-type P. aeruginosa, strain PAO1, LasR acts as a QS regulator, controlling several genes, including those encoding proteases, as well as two other QS regulators, RhlR and PqsR. QS-regulated proteases are secreted by P. aeruginosa into the environment, benefiting the whole population– a cooperative process. When grown in conditions requiring proteases for growth, such as on casein, mutated P. aeruginosa emerge that are capable of using proteases without producing any themselves. While disadvantageous to the community, these “cheaters” experience a fitness advantage. Over time, the proportion of cheaters increase until the population cannot support itself and collapses. Related to QS is the transcription factor MexT, that, when active, down-regulates RhlR. Another transcription factor, MexS, down-regulates MexT expression. In PAO1, MexS is inactive, resulting in constitutively expressed MexT. I was interested in how variants of MexT and MexS that alter expression of QS regulators might influence cooperative behaviors in P. aeruginosa. I compared survival and function of wild type PAO1 to two engineered strains, one with restored MexS function (MexS+) and one with MexT deleted (ΔMexT). I passaged these strains in casein broth for 30 days, evaluating every five days for the emergence of protease-negative mutations. I am also analyzing the genetic composition of each population, focusing on categorizing lasR and a gene involved in peptide transport, psdR. Preliminary results show fewer mutations in psdR than expected in all strains, and fewer mutations in lasR in the ∆mexT strain. Continued exploration of the complex QS and gene regulatory pathways in P. aeruginosa may point to mechanisms of virulence and to potential targets for antibiotic therapies.

The mechanistic target of rapamycin, mTOR, functions in the mTORC1 complex with another protein called raptor as a master regulator of eukaryotic cellular metabolism thereby regulating cell growth including from cancer, cell death including after stroke, inflammation, and aging. In a forward genetic screen for hypoxia resistant mutants, the Crowder lab recently identified a missense mutation in the daf-15 gene, which encodes C. elegans raptor. The mutation produces a heat-sensitive reduction of raptor function, hereafter referred to as daf-15(rf). At 20°C, daf-15(rf) is normally hypoxic sensitive, at 22°C very hypoxia resistant, and at 25°C incapable of normal development. Raptor negatively regulates autophagy, a mechanism for breakdown and recycling of proteins and organelles. Activation of autophagy has been found to promote hypoxic survival in C. elegans and higher organisms. Thus, we hypothesized that activation of autophagy was responsible for the hypoxia resistance of our daf-15(rf) mutant. To test this hypothesis, we first asked whether we could detect increased autophagy using fluorescently-tagged autophagy proteins at 22°C in daf-15(rf) but saw no effect compared to wild type. Next, we asked whether a C. elegans transcription factor, HLH-30, that promotes expression of autophagy proteins was activated by daf-15(rf) and found activation at 25°C but not at 22°C. Finally, we tested whether proteins essential for autophagy were also necessary for the hypoxia resistance of daf-15(rf). By generating double mutant strains, we showed that animals with daf-15(rf) but without essential autophagy proteins were still hypoxia resistant. Thus, we conclude that C. elegans raptor regulates hypoxic sensitivity by an autophagy-independent mechanism. These findings demonstrate that raptor can control hypoxic cellular injury by mechanisms distinct from autophagy. Such mechanisms, if identified, could be targeted for treatment of cancer, stroke, and other diseases where hypoxia plays a role.

mTOR, the mechanistic target of rapamycin, is a serine/threonine kinase that regulates protein synthesis, cell growth, and metabolism in response to nutrients and energy in most eukaryotes. mTOR consists of two distinct complexes, mTORC1 and mTORC2. These complexes can be further divided into three components: mTORC1 consists of mTOR, Raptor, and mLST8, and mTORC2 consists of mTOR, Rictor, and mLST8. mTORC1 is critical in metazoan development and has been implicated in aging, cancer, diabetes, cardiovascular disease, and hypoxia. Previously, the Crowder lab conducted a mutant screen in C. elegans for hypoxia resistant mutations, and identified a missense reduction of function mutation in the daf-15 gene, the C.elegan ortholog of Raptor. A unique feature of this mutation is that the function of Raptor can be turned on and off by varying temperature. It has normal hypoxia resistance at 20 degrees, increased resistance between 21-22, and developmentally arrests at 25 degrees. I and the other authors made use of this conditional developmental arrest phenotype to screen for genetic suppressors. Using genetic mapping, sequencing, and complementation testing, we have identified multiple mutations in three different genes responsible for restoring Raptor function. One of the genes identified in this manner was rnf-126. Results show mutations in rnf-126 suppress the Raptor mutation. A null mutation in rnf-126 similarly suppressed the Raptor mutation. Previous work has implicated mammalian rnf-126 in degradation of the mTORC1 complex in cancer cells, suggesting that reduced levels of daf-15 may produce hypoxia resistance. We tested this hypothesis using auxin-mediated degradation of daf-15, finding that auxin-treated animals are hypoxia resistant. Current work by myself and others will further investigate how rnf-126 controls Raptor function and hypoxia sensitivity. Elaborating the function of this gene will define novel mechanisms whereby Raptor and mTORC1 controls metabolism, hypoxic injury, and development.

Implicit bias, rooted in unconscious attitudes, fuels discrimination based on race, gender, or sexual orientation, disproportionately impacting marginalized groups. Despite the development of interventions addressing provider awareness of implicit bias, the advancement of clinical education through technology has been slow. In the UnBIASED research project, we investigated the usability of ConverSense, a personalized communication assessment tool to raise healthcare providers' awareness of bias in their communication with patients. This web-based tool measures social dimensions such as warmth, interactivity, engagement, and assertiveness from recorded patient-provider visits, and visualizes these patterns through graphs and embedded clips. In this study, we (PT, NE) examined whether ConverSense meets usability standards through heuristic evaluations conducted by design experts. Six healthcare technology experts participated in the evaluation of ConverSense using Nielsen's 10 usability heuristics. Experts documented usability issues for each heuristic and rated their severity on a scale from 0 (not a problem) to 4 (catastrophic problem). Through our analysis, we (PT, NE) identified three cross-cutting themes: 1) Poor design, where experts noted the absence of undo or delete buttons, making navigation challenging, and the distracting color scheme on graphs; 2) Data visualization issues, with experts expressing difficulty interpreting charts and uncertainty about what is considered ideal or good communication. One expert said “It's unclear what is considered ideal/good…for each gauge chart, high interactivity, engagement, and warmth I would assume are ideal/good. But how the charts are displayed in the system I cannot know for certain”; 3) Ambiguity in information presentation, where experts sought more definitions for measured social dimensions and recommended training links to help them identify personal actions they can take to improve their communication. This study underscores the value of incorporating expert feedback and addressing usability issues to improve tools like ConverSense to address implicit bias and promote equitable patient-provider interactions.

Damage to the spinal cord causes one of the most debilitating injuries to the human body. The challenge of promoting the regeneration of this dense network of neurons and glia after spinal cord injury has been seen as insurmountable. However, new techniques emerging from the field of regenerative medicine have illustrated the possibility of encouraging the body to repair these injuries on its own. In the Wills Lab, we study the model organism Xenopus tropicalis, or the Western clawed frog, which has the ability to regenerate its spinal cord and associated tissue following amputation. My project focuses on how X. tropicalis uses the developmental morphogen Sonic Hedgehog (Shh) to re-establish the dorsal-ventral (DV) patterning of the spinal cord during regeneration. I have used cyclopamine, a Shh inhibitor, and SAG, an agonist, in order to perturb Shh signaling during regeneration. I then monitored the effect on DV patterning via immunohistochemical labeling of dorsal and ventral markers. Work so far has shown that Shh signaling is in fact necessary to the establishment of proper DV domains in the regenerate spinal cord. However, my research has also hinted that this specification is complex. Shh appears to have a more proliferative role early on, with patterning effects coming later. In addition, there appears to be an interaction between Shh and other signals that specify anterior-posterior polarity. Overall, my research so far has generated new evidence for how developmental signals are repurposed in the context of regeneration. 

Coccidioidomycosis, also known as Valley Fever (VF) is caused by the fungus Coccidioides. Pigtail macaques (PTMs) bred at the Washington National Primate Research Center (WaNPRC) in Mesa, AZ are naturally infected with Coccidioides and are similar to humans in their physiology, symptoms, and immune responses. Populations with a weakened immune system, notably older individuals, are at risk for severe complications from infection. Additionally, there is evidence that males have a higher incidence of VF than females in endemic areas. I characterized the immune responses in a PTM model across age and sex to better understand how VF affects the immune response of these populations. Forty-two PTMs (2.25-19.24 years, 3.66-18.29 kg, 37 female, 5 male) at the WaNPRC were sampled for blood. The frequencies of immune cell subsets in whole blood were characterized by flow cytometry and compared for significant differences based on age and sex. I analyzed sex-based differences with Brown-Forsythe and Welch ANOVA t-tests and found no statistically significant differences. For age-based differences, we used a simple linear regression to analyze differences by age in immune cell subsets. We found that old PTMs (10.07-19.24 years) have higher activation of CD8+ T cells, myeloid dendritic cells, intermediate monocytes, and higher frequency of γΔ T cells and CD4+ γΔ T cells than young PTMs (2.25-9.69 years). Young PTMs have a higher frequency of CD45+ granulocytes, PD-1 High CD8+ T cells, plasmacytoid dendritic cells, and NK cells. By correlating older PTMs with higher immune cell activation, and younger PTMs with higher immune cell frequency, we have a better understanding of how a vaccine or treatment could be developed to support older individuals, who are at greater risk of severe infection.

Corepressors are proteins recruited by partner proteins to negatively influence the transcription of genes. TPL is a corepressor from the model plant Arabidopsis thaliana, and while we understand a lot about how TPL works, many mysteries still remain. My project aims to identify other proteins that work with TPL to form a transcriptional repression complex at a single-engineered promoter site. First, we created a synthetic repressor called dCas9-TPL that binds and represses the transcription of the RUBY reporter. The RUBY reporter is a visual marker designed to express throughout the entire plant, turning the green plant a bright purple. Our engineered RUBY line also carries two guide RNA binding sites in its promoter with sequences not found anywhere else in the Arabidopsis genome. This allows dCas9-TPL to bind to and repress this synthetic gene and not affect the transcription of other genes. Many of these plants have morphological phenotypes, and visual screening of the repressed RUBY line showed the plants turn a faint whitish-pink instead of bright purple, signifying that the repression by TPL is working. I have screened mutagenized populations of 40,000 individuals from the validated repressed RUBY plant strains using the Ethyl methanesulfonate (EMS) protocol, which creates new point mutations. I identified 257 individuals from 129 mutagenized families with bright purple organs, which signifies that the RUBY reporter is no longer repressing due to a putative TPL interactor being mutated. I will then proceed to form complementation groups and subsequent DNA sequencing to map the mutations. By identifying regulators of corepressor function in plant biology through downstream whole genome sequencing, I hope to learn principles that can inform cellular engineering across many organisms and better understand why certain mutations associated with transcriptional repression cause developmental defects or diseases like cancer in humans.

Social interactions in humans have shown to improve pain outcomes and diminish the development of mechanical hypersensitivity (allodynia) following injury. This effect is known as social buffering of pain; however, the underlying mechanisms are not well understood. Prior preclinical studies focused on forced social interactions between unfamiliar mice, lacking translational value to patients. To fill this gap, our research explores how volitional social behavior shifts pain sensitivity and affect following a neuropathic injury. Volitional interaction is key to socialization as individuals usually socialize because they want to, not due to force, which makes studying how mice voluntarily interact with each other important. To determine how volitional social interaction impacts both sensory and affective (emotional) components of pain, we use male and female mice who have received a spared nerve injury (SNI). Trained in social self-administration, mice learn to lever-press to engage with a familiar conspecific. Mice are then tested in von Frey where thin plastic filaments of increasing weights are applied to the mouse hind paw before and after SNI. These filaments do not cause pain, rather elicit a pain response of withdrawing the paw. To determine sensory sensitivity, the weight when the animal's paw is withdrawn is recorded as percent change from baseline. To determine changes in affective pain, the amount of time the animals hold their paw up, following withdrawal, is recorded as percent change from baseline. We found that male and female mice show significant attenuation in their mechanical hypersensitivity following volitional social interaction compared to mice deprived of volitional social interaction. Males show even less mechanical sensitivity, indicating that males may be more impacted by social analgesia than females. Understanding the divergent responses between male and female mice and the role of volitional social interaction in pain modulation, offers potential avenues for developing novel therapeutic strategies.

Chemical communication is the oldest and most widespread form of communication across the tree of life, and markedly present among lizards. However, the drivers of chemical profile variations in this group remain for the most part uninvestigated. In South American lizards of the Tropiduridae family, semiochemicals are produced by epidermal gland organs called α-glands, exclusively found on the ventral side of male individuals of at least 40 species from four genera. The chemicals produced by these glands are hypothesized to interact with their environments in different ways since chemical species are naturally reactive and tend towards their lowest energetic state. Thus, the intrinsic properties of a semiochemical impact its survival and efficacy for communication. Given the diverse ecology and broad geographical distribution of tropidurids, we investigated whether variation in the chemical composition of α-gland secretions correlates with temperature, humidity, and habitat openness. We performed liquid chromatography-mass spectrometry (LCMS) to obtain the metabolomes of three different sample types. We sampled male skin containing the α-glands, undifferentiated male skin, and female skin. Environmental and chemical property data were extracted from online databases, literature, and field observations. Preliminary tests were done by making Venn diagrams comparing the metabolomes of each sample type. These revealed differences in metabolite compositions, notably between males and females as well as between glandular and undifferentiated skin. From the metabolomes of α-glands, we expect to see chemical species with properties that confer greater survival given the specificities of the environment. For example, given a lizard from a hot and humid environment, we expect the metabolome of the α-glands to contain higher molecular weight species with less functional group complexity. Understanding how environmental parameters drive the chemical composition of α-glands is expected to provide a deeper understanding of the evolutionary history of chemical signaling in terrestrial vertebrates.

Traumatic spinal cord injury (tSCI) is a devastating condition that causes sensory and motor dysfunction and permanently impairs normal life. Spasticity is one of the most common complications associated with tSCI that limits independent functional abilities. Spasticity is defined as a velocity-dependent increase in muscle tone, in response to passive movement and it is accompanied by pain and stiffness. Unfortunately, current treatments provide only transient and often incomplete relief of spasticity and individuals often experience long-term adverse effects. Through a collaborative project between three labs, we aim to develop a durable non-invasive electrical stimulation treatment to alleviate spasticity. I participated in preparing the model of spasticity by performing spinal surgeries on the cervical spine of rats. To evaluate spasticity, we studied the loss of Rate-Dependent Depression (RDD) of the H-reflex which is considered the electrophysiological hallmark of spasticity. To do so, I fabricated an electrode nerve cuff that was implanted on the median nerve of the rodent’s forearm to study the H-reflex of the affected muscle in the rat’s forelimb. I then recorded and analyzed the temporal development and change of spasticity. H-reflex results validated the spasticity model by showing RDD reduction in injured rats compared to the uninjured rats. The developed treatment shows promising modulation of the H-reflex and recovery of RDD in injured animals. Additionally, to measure velocity-dependent muscle tone, we developed a robotic device that passively moves the rodent’s forearm at different velocities. Employing this robotic behavioral assessment allows me to objectively assess the effect of stimulation on spasticity in the rodent forelimb. Obtained data reveals the muscle resistance to be three times higher in the injured rodent. This novel therapeutic stimulation protocol could potentially be used for clinical use as a non-invasive therapy, to help patients with spasticity in the hand after suffering from cervical tSCI.

The effective treatment of individuals with HIV relies on maintaining therapeutic drug concentrations, necessitating accurate measurement of antiretroviral (ARV) drug levels. Current methods, such as liquid chromatography tandem mass spectrometry (LC-MS/MS), are limited by cost and accessibility. Our research addresses this gap by developing the INTEGRase activITY (INTEGRITY) assay for measuring integrase strand transfer inhibitors (INSTIs), a leading class of ARV drugs. This 2-step assay quantifies INSTIs using a DNA strand transfer reaction and quantitative polymerase chain reaction (qPCR). The presence of INSTI drugs disrupts the strand transfer reaction, inhibiting full-length target DNA formation, which is then measured through real-time qPCR. My work focused on optimizing the limit of detection of INTEGRITY by altering the strand transfer reaction conditions and protocol. Specifically, I conducted experiments altering INSTI drug concentrations and optimizing pre-incubation times of integrase with the drug to enhance the LOD. I observed that preliminary incubation of integrase and INSTI drugs for 5 minutes at 37 degrees Celsius improved the LOD of INTEGRITY by an order of magnitude. The simplicity of the INTEGRITY assay, utilizing standard laboratory equipment, holds immense promise for broadening access to routine clinic-based ARV drug level monitoring. This advancement has the potential to significantly enhance HIV care on a global scale by offering a cost-effective and accessible solution for monitoring therapeutic drug concentrations.

The antibiotic penicillin is highly effective at treating the STI syphilis, caused by the bacterium T. pallidum. However, the United States has seen increases in syphilis cases every year for the past 20 years; congenital syphilis cases have risen more than 219% from 2017 to 2021 and overall syphilis cases have risen 32% from 2020 to 2021. This situation demonstrates the need for an effective vaccine as current approaches are not working. The aim of this project is to utilize phage immunoprecipitation sequencing (PhIP-Seq) techniques to assist in the development of an effective vaccine in rabbits and eventually humans. To this end I have been using PhIP-Seq techniques to systematically profile the immune responses to vaccine candidates and T. pallidum infections in rabbits. When rabbits are immunized with a cocktail of three strains of the protein TprC we saw a protective immune response against treponemes (resulting in no viable treponemes) whereas an immunization with TprD saw reduced immune protection. I used PhIP-Seq methods - informed by next-generation sequencing (NGS) and differential expression analysis - to determine the epitope-specificity of antibodies in polyclonal serum samples from rabbits immunized with these vaccine candidates. Epitope-specificity comparisons between the resulting antibodies of the two immunogens can shed light on regions of these proteins critical for protection against treponemes. In the next few months I plan to integrate alanine scanning mutagenesis into the project to assess amino acid binding specificity and accurately identify crucial residues for antibody-binding. The fusion of scanning mutagenesis with PhIP-Seq will allow me and the other research scientists assisting with the project to refine of the effectiveness of our existing vaccine candidates.

CD-1 outbred mice are a preferred mouse model used in behavioral research due to their genetic diversity and overall capacity for acquiring learned behavior. Psychiatric traits involve cognitive brain function, and psychiatric disorders always result in cognitive dysfunction. Therefore, behavioral research aimed to model psychiatric disease states can maximize translational value to human brain function and behavior by incorporating the study of effects on cognition in CD-1 mice, specifically. However, behavioral tests that target cognition sensitivity have been only made in other mouse strains. Here, we present a study establishing a human-compatible touchscreen-testing environment where we conduct the 5-choice Serial Reaction Time (5-CSRT) task, a mouse-friendly adaptation of the human continuous performance task on CD-1 mice from both sexes to phenotype baseline attention, impulsivity, and motivation. The 5-CSRT task is administered within automated Bussey touchscreen chambers and requires extensive behavioral shaping, habituation, tone, and screen touch training sessions designed to activate underlying neural circuitry involved in cognitive function. Before moving on to the next session, specific criteria must be met in order to promote acquired learning. These sessions become progressively more difficult, with an ultimate goal of priming the mice for the 5-CSRT touchscreen task, whereby animals need to find and touch a visual target being displayed from 5 available options on a touchscreen within a given amount of time to receive a food reward. To analyze and phenotype cognition, we combined the use of camera recordings to observe high-resolution behavioral dynamics in addition to summary task metrics that include accuracy, omissions, premature responses, and food-retrieval latency. Our results establish the use of CD-1 mice with human-compatible touchscreen-based approaches to facilitate an entry point in the effective modeling of psychiatric disorders that can map to human cognitive dysfunction.

Maladaptive aggression characterizes - or is comorbid with - many neuropsychiatric illnesses, and can have devastating effects on individuals, their caretakers, and healthcare professionals. Human aggression is typically demarcated as exhibiting either reactive (defensive) or appetitive (rewarding) components. Despite a significant clinical awareness of the differences between these aggression presentations, preclinical characterization of their relative circuitry and associated neuronal mechanisms are absent. Using recently established protocols within our lab, we are able to study and compare these aggression phenotypes in outbred male mice in a high throughput manner. Briefly, for appetitive aggression, we train mice to self-administer a novel subordinate intruder over 7 days using a trial design. In the reactive condition, we non-contingently administered intruders with the same frequency distribution as the appetitive mice. In the current experiment, we used CD1xVgat-Cre mice injected with pGP-AAV-syn-FLEX-jGCaMP7s in the rostral lateral septum (LSr) to examine cell-type specific activity via fiber photometry. GABAergic activity in the lateral septum has historically been implicated in the control of reactive aggression, but its role in appetitive aggression is unknown. My roles in this project include behavioral testing and filming of the mice, as well as scoring these videos for first attacks following intruder presentation. Using these timestamps, I will next analyze the changes in population level dynamics across different time points of aggression motivation, seeking, and consumption using the open source photometry analysis program GuPPy. We expect that the photometry results for mice in reactive and appetitive environments will show different patterns of activity, with more GABAergic activity during the consumption of reactive aggression. Interestingly, our preliminary results also show an increase in GABAergic activity when mice press the lever for a trial on which they subsequently attack, indicating that GABAergic activity may drive appetitive aggression seeking.

General anesthesia (GA) is administered as a sedative in nearly 60,000 surgeries daily in the United States. Yet, there is a very limited understanding about how GA impacts brain activity, leading to induced loss of consciousness and pain sensation. Preliminary work in the Heshmati lab has highlighted key subcortical structures that are engaged during anesthesia, but it remains unclear how activity in these regions and across the brain regulates awareness or pain sensation as anesthesia is induced (“induction”), maintained at a steady state (“maintenance”) and removed (“emergence”), as is done during surgeries. My work aims to identify the neural circuits that regulate the loss of consciousness and pain sensation during GA by recording local field potentials (LFP) from mice as they undergo volatile anesthetic isoflurane (ISO). During LFP recordings, I will insert small electrodes into highlighted regions of interest, to capture low-frequency extracellular voltage signals generated by the synchronized activity of nearby neural populations during the three periods of interest: induction, maintenance, and emergence from isoflurane GA. I will analyze the amplitude fluctuations and frequency patterns to identify synchronized oscillations within subregions and assess the level of synchrony, or coherence, across different regions. Given previous findings on the shared and opposed involvement of subcortical regions in pain and anesthesia, I expect to observe coherence among some of the regions, such as the amygdala and hypothalamus, but potentially anti-correlation within specific subsections, such as central vs. basolateral amygdala. Through these experiments, I will be able to monitor the effects of isoflurane anesthesia through a temporally-defined electrophysiological lens, capturing real-time activation dynamics of large neural populations across induction and recovery from anesthesia. Thus, my research aims to further develop our understanding of the brain under GA, by providing novel insight into the neural circuits regulating wakefulness and pain during surgical procedures.

Neuropsychiatric disorders, such as major depressive disorder, pose a difficult challenge for healthcare providers. Treatments for such disorders vary in efficacy and come with detrimental costs. Historically, preclinical animal models have failed to incorporate the nuances of volitional human social behavior. This project used chronic social defeat stress in which mice experienced bouts of antagonistic encounters to induce depression-like behaviors in male and female mice, this was followed by self-administered social interactions within an experimental chamber in which lever presses were reinforced by social contact. The goal is to develop preclinical animal models that can be assessed to identify neural mechanisms responsible for stress-induced social motivation. Male and female mice will train to self-administer social interaction with a sex and age-matched housing partner over the course of ten 12-trial sessions. Next, experimental male and female mice will be subjected to both social and witness defeat (observation of social defeat) sessions followed by social self-administration. Before and after the 10-day social stress sessions, we will test social reward seeking via non-reinforced self-administration of social reward followed by a progressive ratio test. Brain tissue will be collected and prepared for immunohistochemistry and whole-brain clearing. Social defeat decreased social reward seeking behaviors in male mice. Witness defeat did not alter social reward seeking in males but increased seeking behavior in female mice. Social stress can be used to discern differences in social motivation in male and female mice as a result of stress-induced factors. There is potential in using whole-brain activity mapping to identify brain structures activated during social reward following social stress. We hope to build a technical tool for the field that can encompass whole-brain activity responsible for social stress responses by utilizing nuclear localization and retrograde tracing.

Dopamine (DA) producing neurons of the ventral tegmental area (VTA) in the midbrain regulate reward association learning and motivation. These DA neurons are modulated by neuropeptides and can be separated into distinct subpopulations based on differential gene expression, regulation of activity, and projection patterns. But how these different patterns are established and contribute to distinct functions of DA subpopulations remain poorly understood. One potential key component for these neuropeptides is the transient receptor potential canonical (TRPC) channels. Specifically, we identified the gene encoding TRPC type 6 channel (Trpc6) as having enriched expression in the VTA DA neurons. To determine whether Trpc6 is differentially expressed in VTA DA subpopulations, I utilized the quantitative, multiplexed in situ hybridization methods. Using wild-type mice, I probed for the expression of tyrosine hydroxylase (Th), a marker of all DA neurons, and Trpc6 as well as markers of two subpopulations, corticotropin releasing hormone receptor 1 (Crhr1) and cholecystokinin (Cck). The analysis showed that Trpc6 expression is significantly higher in the Crhr1 subpopulation, of 81%, than in the Cck subpopulation, of 66%. Because neuropeptides like neurotensin increase calcium concentration in DA neurons, we hypothesized that TRPC6 contributes to these neuropeptide-evoked calcium signals. To investigate the role of TRPC6 in DA signaling, I used a viral-based CRISPR/Cas9 approach to induce selective mutagenesis of TRPC6 in specific DA subpopulations. Then, I assessed the calcium responses of subpopulations to neurotensin by measuring the amplitude and neurotensin-evoked oscillations using acute brain slices. We expect the calcium responses to decrease more in the Crhr1 subpopulation than in the Cck subpopulation compared to the control as the Crhr1 population has higher Trpc6 expression. By elucidating the role of TRPC6, we hope to contribute to discovering pharmacological interventions for diseases caused by dopaminergic system dysfunctions such as Parkinson’s disease and substance use disorders.

Lizards in the family Tropiduridae have ventral epidermal gland organs that are involved in chemical signaling and whose secretory mechanism is entirely unknown. This is because, like other epidermal generation glands, 'alpha-glands' lack a pore through which their secretion can be exerted. Chemical signaling is a valuable aspect of tropidurid lizards' social and ecological interactions, and some have been observed territorially scraping their alpha-glands against the substrate. This process has been hypothesized to facilitate the release of chemical signals via abrasion. To investigate this abrasion hypothesis, we analyzed 74 skin samples from 27 tropidurid species, using light microscopy and scanning electron microscopy (SEM). The SEM revealed incredible surface variability in epidermal glands, providing morphological insight. We found that the exposed glandular mass of each gland scale rests atop the oberhautchen layer of the skin's subjacent generation, which indicates that the secretion of chemicals involves exposing a mostly solid glandular material on the outside of the scales. Histological sectioning of gland scales revealed morphological consistency, indicating that the same secretory mechanism is shared across the tropidurid phylogeny. Imaging of histology samples also revealed that the shedding process which exposes the glandular material may be facilitated by the clear layer, found directly above the glandular mass during development. Characterization of morphological patterns in the formatted SEM images and comparison with histological data should provide evidence for or against taxon-specific or ecology-specific alpha-gland structures, and further support the idea of chemical secretion requiring epidermal exposal of glandular material. Investigations of the morphology and functional mechanism of this unique organ provide insight into the behavior and evolution of tropidurid lizards and shed light on factors influencing the evolution of chemical signaling in terrestrial organisms. 

The Pacific Northwest (PNW) saw an unprecedented heatwave between June 25 to July 3 of 2021, with temperatures reaching up to 15℃ above the climatological mean. Previous research has examined the impact of this event on plants in Western Washington and Oregon through observational studies, and has focused on the economic implications for poor crop turnout. We used remote sensing data to take a top-down approach and examined how all plants throughout the PNW fared during and after this historical heatwave. Solar induced fluorescence (SIF) and Near-Infrared Reflectance of vegetation (NIRv) are two remotely sensed products that have been used to estimate plant health and gross primary productivity (GPP). SIF is more closely connected to plant processes like photosynthesis but has a short record (2018-2021) compared to VIIRS NIRv (2012-2021). We compared the responses of SIF to NIRv and found that both vegetation indices increased in trees and woody savannas, but decreased in grasslands and crops. However, SIF showed more intense and geographically larger increases in areas covered by trees. We then compared these vegetation indices to in-situ flux tower measurements of carbon fluxes, which did not always agree with SIF during the heatwave in woody areas. This study shows how remote sensing can further our understanding of how extreme events impact plant health, which is increasingly important as heatwaves become more intense and frequent in the future.

Sudden unexpected death in epilepsy (SUDEP) is the most severe consequence of uncontrolled epilepsy. SUDEP is a multifactorial disease associated with serotonin (5-HT) imbalance and exacerbated neuroinflammation. Unfortunately, current preclinical animal models do not adequately explain all underlying causes of these events or their subsequent effects. Seizures are a common comorbidity in Alzheimer’s disease (AD), especially in patients with genetic variants in amyloid precursor protein (APP) and presenilin 1 (PSEN1) and 2 (PSEN 2). Clinical evidence suggests that seizures in AD patients worsen their cognitive decline and increase mortality rate compared to AD patients without seizures. Our lab demonstrated that 2-month-old mice with an APP/PS1 variant subjected to chronic evoked seizures resulted in premature mortality, heightened neuroinflammation, and altered 5-HT system enzyme expression prior to AD onset. These findings reveal a novel preclinical platform to test potential preventative agents for SUDEP. Given the relationship between seizures and AD, I aim to prevent seizure-induced premature mortality, and define 5-HT and neuroinflammatory changes in APP/PS1 mice treated with 2 investigational agents: lorcaserin, a selective 5-HT receptor agonist, and cannabidiol (CBD), a broad-spectrum anti-inflammatory and 5-HT modulator. I hypothesize that targeting seizure-induced neuroinflammation and the dysregulated 5-HT system with these compounds will decrease premature seizure-induced mortality. To assess this, 2-month-old APP/PS1 mice underwent corneal kindling procedure to evoke investigator-controlled chronic seizures and received lorcaserin (10 mg/kg) or CBD (100 mg/kg) via the intraperitoneal route. Then, I tracked survival during the chronic seizure period and performed molecular analysis to quantify neuroinflammatory proteins and 5-HT system enzyme expression. Our preliminary results show that mice treated with lorcaserin or CBD had a mortality rate of 10% compared to 75% in the untreated APP/PS1 mice. Future directions include using these compounds in other preclinical SUDEP models to confirm the translational potential of these medications to clinical use.

Repetitive transcranial magnetic stimulation (rTMS) is a psychiatric treatment which has shown promise for experimental treatment of memory loss in Alzheimer’s Disease. rTMS uses a coil and electric current which is able to create a magnetic field that can depolarize neurons noninvasively and induce synchronized activity of large populations of neurons, ultimately inducing, lasting changes through synaptic plasticity. Alzheimer’s disease patients show disruptions in the Default Mode Network (DMN), a network of brain regions which is typically active at rest. The DMN has an important role in memory consolidation and is disrupted in Alzheimer's Disease. We hypothesize that strengthening the default mode network through rTMS applied to area left 8AV of the frontal cortex will create improvements in patient memory. To answer this question, we are performing a single-blind, single-arm, randomized cross-over trial of rTMS on early-stage Alzheimer's disease patients. Region 8AV is located by using MRI scans obtained before patients receive either the sham or experimental procedure. This region was chosen due to its connection to the default mode network and previous promising TMS research. Our primary outcome measure is the speed of forgetting, a new, reliable index of memory function obtained by fitting a computational model of episodic memory to behavioral data from an adaptive memory test. Due to the frequent use of rTMS in mood disorder treatment, we are using depression and anxiety scales to track possible mood improvements as a secondary outcome measure. MRI scans will also be analyzed to see if the experimental treatment caused any structural differences in patient brains. Should our hypothesis be correct, we expect to see improvements in memory or cessation of memory decline in patients. Successful treatment would provide a novel target for Alzheimer’s Disease treatment using rTMS, and additional evidence for the continued investigation of rTMS for Alzheimer’s Disease.

 The Amazon rainforest is one of the most biodiverse regions on this planet and home to thousands of species of butterflies. Our pilot study aimed to investigate correlations between vegetation diversity and temperature and butterfly species richness in the Madre de Dios region of southeastern Peru. This study was conducted across three sites of differing plant diversity, including a monoculture orchard, a mixed orchard, and a rainforest. We hypothesized that there would be a strong positive correlation between species richness and vegetation diversity, resulting in high species richness in the rainforest. We also predicted that each species would only be found within one site, as most butterflies rely on specific plant species for reproductive purposes. Finally, we hypothesized that the hottest times of day would have the least amount of butterfly activity. By attracting butterflies with baited banana leaves, we were able to record an array of species during multiple, daily 45 minute observational sessions. After classifying photographs of butterflies during our sessions to morphospecies and averaging species richness, we found that the sites with higher vegetation diversity had a wider range of species. Most species demonstrated specialist behavior, found only in one site. Out of 26 morphological species, only three were observed at more than one site. Limitations in our sample size made any data surrounding behavior across a temperature gradient inconclusive. By critiquing this pilot study and analyzing its small set of data, we suggest protocols for future research on butterfly species richness and activity.

Apolipoprotein E (APOE) is a polymorphic gene with 3 main alleles: APOE2, APOE3, APOE4.The APOE4 allele is the greatest genetic risk factor of late-onset Alzheimer’s disease (AD). Carriage of the APOE4 allele results in increased amyloid-β (Aβ) accumulation, aggregation, and deposition in the brain compared to other genotypes, thus contributing to AD pathogenesis. Preeclampsia (PE) is a pregnancy-specific disorder associated with maternal and perinatal mortality. PE has also been linked to protein misfolding pathology, characterized by the accumulation of misfolded proteins (including Aβ) in the placenta, urine, and blood. Due to the similarities with AD pathology, we investigated the relationship between APOE4 and the development of PE-symptomatology in a transgenic mouse model carrying human APOE. To assess this relationship, we induced a PE-like syndrome in APOE3 +/+ and APOE4 +/+ pregnant females on gestational day 13.5 using the Reduced Uteroplacental Perfusion Pressure (RUPP) procedure (n=5-6/group). Sham procedures were performed on the same number of females in each group as controls. On gestational day 17.5, differences in blood pressure, proteinuria, kidney integrity, placental efficiency, placental morphology, and fetal weights in response to RUPP were assessed and compared between APOE3 and APOE4 mice. Our results show that RUPP induced significant kidney damage in APOE4 mice (p=0.014), while APOE3 mice maintained normal kidney morphology. Moreover, RUPP was associated with a significant reduction in placental efficiency in both groups, but only APOE4 fetuses developed fetal growth restriction (FGR, p=0.045). APOE3 was also associated with mild changes in placental morphology, but APOE4 was not. Blood pressure or proteinuria did not vary in response to RUPP in either genotype. Our findings suggest that APOE4 confers an increased susceptibility to kidney damage and FGR in response to RUPP compared to APOE3. Further investigation is needed to determine the molecular basis of these genotype-dependent effects.

By 2050, it is predicted that over half of the global population will be over the age of 65. Therefore, it is of the utmost importance that we study the impacts of aging on human health and well-being. One major obstacle researchers face in seeking to understand human health relates to the extensive number of newly discovered biological systems that exist and interact in every individual at various levels and timescales. Furthermore, many well-known age-related neurodegenerative diseases are thought to begin years to decades before any clinically relevant symptoms are present. The current study attempts to address some of these challenges by examining the interaction of cognition with immune function, inflammation, gene expression, and the microbiome. This research will allow us to begin unpacking the complex interactions of these numerous biological factors and their impact on natural aging and age-related neurodegenerative diseases. To accomplish these goals, our project has created and implemented a novel high-throughput cognitive testing system to assess a range of cognitive abilities including, but not limited to, attention, memory, and object recognition, on two species of well-known non-human primates (NHPs). At numerous time points, we concurrently collected and analyzed blood, fecal, and cerebrospinal fluid to characterize each animal's health profile. With our collection of data sets, we anticipate that older NHPs will display higher levels of neuroinflammation and decreased immune function. We hope to find correlations between these factors and other variables with genes that are known to be related to a host of neurodegenerative diseases including Alzheimer's Disease (AD), Alzheimer’s Disease-Related Dementia (ADRD), and Parkinson’s Disease, among others. Results from this ongoing project will unravel mechanisms associated with age-related neurodegenerative diseases, allowing for earlier detection; this early detection is regarded as the most effective approach for preventing and treating such diseases.

Mind-wandering, characterized by the inward direction of attention towards thoughts, represents a widespread cognitive phenomenon that can disrupt task-related attention. Evidence indicates that the frequency of mind-wandering increases during sustained attention tasks, and previous research has proposed several hypotheses, such as resource-depletion, control-failure, and resource-control hypothesis, to explain the increases as well as the declines in task performance from their perspective. This study first aims to investigate the impact of incorporating a strategy of break on the frequency of mind-wandering during a Sustained Attention to Response Task (SART). It seeks to determine whether a break strategy can mitigate the frequency of mind-wandering and to identify which of the aforementioned hypotheses best fit the experiment outcome. Specifically, it examines whether a rest break or a task-switching intervention can reduce the frequency of mind-wandering, comparing these effects to a no-break condition. Forty students from the University of Washington will participate in a four-part experiment that includes two 480-trial SART tasks, an intervening break condition (rest break, task-switch, or no break), and a concluding questionnaire. Mind-wandering will be measured through thought-probe methodology, and task performance will be assessed using error rates and response times. Additionally, subjective workload and fatigue level will be assessed using NASA Task Load Index (NASA-TLX) questionnaire. Based on the three existing hypotheses and behavioral data, the second aim of the study is to construct three cognitive architecture ACT-R models, and by comparing different models to reflect potential mechanisms of the role of breaks in mind-wandering.

The potential cognitive benefits of mindfulness meditation, such as its effects on memory processes, are gaining increasing attention. The growing interest in mindfulness meditation as a technique to improve cognitive functions, particularly long-term memory, motivates this investigation. The purpose of this study is to determine how mindfulness meditation affects the individual’s rate of forgetting in long-term memory as practice days increase. Using a computational model of episodic memory fitted to data obtained from an adaptive memory test, the study is able to monitor and predict the rate of forgetting for each individual and acquire a deeper understanding of how mindfulness meditation influences individual differences in memory retention and retrieval. The study will recruit thirty healthy participants through the Psychology Subject Pool of the University of Washington. The participants will be randomly assigned to either a group that practices mindfulness meditation or a control group that engages in podcast listening. For six days, participants will complete online memory tasks before and after each mindfulness meditation session or podcast listening session, while their performance data will be analyzed to model the rate of forgetting. To assess the principal effects of mindfulness meditation on the rate of forgetting, statistical analyses, such as linear mixed models, will be employed. We hypothesized that participants who participate in mindfulness meditation will have a lower rate of forgetting than those in the control group who engage in podcast listening after six days. Moreover, the study also hypothesizes that participants engaging in mindfulness meditation will demonstrate a decreasing rate of forgetting as the number of practice days increases. The finding of this study may inform interventions using mindfulness to enhance memory retention, benefiting those with age-related cognitive decline or stress-related memory challenges. It contributes to understanding the potential of mindfulness in cognitive function and memory-related disorders.

Angiogenesis, or the formation of new blood vessels, is crucial for normal bodily function but is especially important in diseases that cause blood vessel breakdown such as diabetic vasculopathy. Angiogenesis is regulated by activation of the Tie2 receptors in endothelial cells, which have two main ligands: angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2). Ang1 binding has been shown to stabilize blood vessels and inhibit vascular leakage, while Ang2 antagonizes these effects. We have previously shown that a computationally designed Tie2 super-agonist which presents eight copies of the Ang1 F-domain strongly activates Ang1-like signaling in human umbilical vascular endothelial cells (HUVECs). In this project, we hope to assess the Tie2 super-agonist’s ability to rescue diabetes induced blood vessel defects in a diabetic blood vessel organoid (BVO) model. To model diabetic conditions, a three-dimensional blood vessel organoid model has been cultured in a high glucose media along with inflammatory cytokines associated with the diabetic phenotype. Western blotting and immunofluorescence staining will be used to assess the relative quantities and localization of proteins involved in vascular stability and inflammations upon treatment with the Tie2 super-agonist. Vascular degeneration is a very harmful condition associated with many prevalent diseases including diabetes, so the Tie2 super-agonist could potentially be a new therapeutic drug candidate for treating blood vessel dysfunction in patients with these conditions in the future.

Alzheimer's Disease (AD) impacts over 6 million people in the U.S, but there are currently no fully effective treatments. Ageing is the biggest risk factor for AD and is associated with cellular changes called senescence. Cellular senescence describes a natural process in cells, leading to cell cycle arrest and metabolic changes due to insults from aging/disease processes. Factors contributing to senescence include DNA damage, and others. A risk factor in neurodegeneration is the ageing of microglia- our brain's immune cells that maintain a healthy brain. Senescent microglia express a senescence associated secretory phenotype- a combination of inflammatory proteins released into their environment- that enhances neurodegenerative processes. My project investigates the relationship between microglial senescence and AD by comparing the levels of senescence markers in AD brains, healthy young brains, and aged brains. I hypothesized that AD brains will contain the greatest amount of senescence markers, followed by aged brains, then healthy young brains. I performed immunohistochemistry for p16Ink4a and gammaH2AX (two robust senescence markers) on 10 human individuals (5 male/5 female per cohort) who donated their brain post-mortem. p16Ink4a is involved in cell cycle regulation and gammaH2AX signals DNA damage. The brain samples were also stained with Iba-1 to identify microglia. A confocal microscope imaged the samples and data was analyzed using the IMARIS software and ImageJ. Senescence markers were quantified in each cohort and localized in microglia or non-microglia cells. I expect to see the greatest amount of p16Ink4a and gammaH2AX in AD brains (specifically AD microglia), with the least amount in healthy young brains. I also expect co-localization of gammaH2AX and p16Ink4a in my samples. Understanding the relationship between microglial senescence and AD pathology could aid in finding methods to target cellular senescence. Slowing down this process could be a usefull tool in decreasing the progression of AD.

The Campanian stage of the Cretaceous (~84–72 million years [Ma]) was the zenith of dinosaur diversity. Western North America is highly fossiliferous and preserves Campanian-age rock units throughout the Western Interior Basin. Most studies that investigated dinosaur diversity from this interval used data obtained from macrosites (e.g., skeletons), whereas few have investigated vertebrate microfossil sites. Vertebrate microfossil sites are a rich source of data on biodiversity (e.g., taxon richness, relative abundance) and how it changes through time. The Judith River Formation of north-central Montana is rich in vertebrate microfossil sites, preserving 4 million years of the Campanian (~79–74 Ma). Here we aim to observe patterns of dinosaur diversity in the Judith River Formation by quantifying dinosaur taxon richness and relative abundances based on dinosaur teeth from two stratigraphically and temporally separated microfossil sites. These sites are the lower Makela-French 1 (~77 Ma) and the upper Clamfetti (~75 Ma). Presently, we have 300 specimens out of a planned 400. We hypothesize that changes in diversity and abudance occurred between these two sites. Our preliminary results reveal a change in dinosaur diversity between Makela-French 1 and Clamfetti. Hadrosaurs and ceratopsians are present and relatively abundance at both sites, whereas ankylosaurs decrease in abundance from Makela-French 1 to Clamfetti. Small herbivores like pachycephalosaurs and hypsilophodonts are rare at both sites. Theropods show similar patterns to the herbivore’s trends. Tyrannosaurs and dromaeosaurs are common at both sites, whereas troodontids are absent from Makela-French 1. These preliminary findings reflect diversity patterns that are not easily observable solely through the collection of dinosaur macrofossils. Our continued collection of fossils from Makela-French 1, Clamfetti, and additional sites will increase our sample size and provide better fine-scale resolution of dinosaur diversity patterns during this crucial interval in their evolution.

The climate crisis is predicted to increase the occurrence of extreme weather events such as heat waves and polar fronts, which will disproportionately affect vulnerable populations such as people experiencing homelessness (PEH). As climate change increases social and economic instability, policymakers need to be informed on how to effectively protect the interests of our most vulnerable populations. Building off of studies that have analyzed the impact of heat and cold on hospitalizations among PEH, I examine the health effect of extreme weather events in King County, San Francisco County, and Los Angeles County. Temporary emergency shelters are one of the main strategies used to protect the wellbeing of the PEH population during extreme weather events. Moreover, I intend to determine the effectiveness of emergency shelters in reducing the impact of extreme weather on the death toll and/or health complications of PEH. Utilizing data on health impacts among PEH, daily maximum/minimum temperatures, and cooling shelters, I will use regression models to determine the relationship between maximum/minimum daily temperature and health outcomes and if cooling shelters reduce negative health impacts during extreme weather events. I expect to find a positive relationship between extreme temperatures and hospitalizations, as prior studies have observed, but that this relationship will be weaker on days on which cities open emergency shelters. However, it is plausible that because emergency shelters have restrictions on access, limited bed capacity, or inadequate public outreach, the impact of emergency shelters on hospitalizations may be insubstantial. My results will contribute to a growing body of literature that points to a relationship between extreme weather and poor health outcomes for PEH. This evidence will have implications for the importance of allocating funding towards the shelter system and other social service efforts, while clarifying the effectiveness of the emergency shelter system.

Bacterial CRISPR immune systems defend against foreign genetic material, such as bacteriophage viruses. CRISPR systems are classified into six types with diverse protein components and mechanisms of interference. Among these, our research investigates the function of CRISPR-Cas13 systems, which uniquely target RNA rather than DNA. To overcome immunity, bacteriophages have evolved anti-CRISPR mechanisms that are designed to inhibit specific CRISPR types, restoring infection and proliferation of the viral invader. We recently discovered a novel anti-CRISPR mechanism, in which a noncoding RNA provides inhibition of CRISPR-Cas13 function. The central questions surrounding this RNA anti-CRISPR (rnAcr) are how it associates with CRISPR-Cas13 in order to inhibit its function, as well as the boundaries of its length and anticipated mechanism of inhibition. rnAcr is predicted to have three vital stem loops, which have been experimentally deleted and structurally disrupted by performing site directed mutagenesis to mutate select regions of nucleotides in each stem’s structure. We did this in order to determine if the stem loops’ structures were necessary for rnAcr’s anti-CRISPR function. We found that these were all essential for its function, which gives rise to the hypothesis that its structure is interacting with the bacterial host’s CRISPR-Cas13 system to effectuate its inhibitory mechanism. In order to test anti-CRISPR function, we conjugated a target and nontarget plasmid, in which the target plasmid would be recognized by Cas13, and cellular RNA would be cleaved, leaving no growth if no anti-CRISPR mechanism is present. We have shown that rnAcr is sufficient for anti-CRISPR function, allowing for tolerance of these target plasmids and cellular growth. rnAcr suggests a novel anti-CRISPR mechanism, as until now, the majority of reported anti-CRISPRs have been composed of small proteins produced during phage infection, suggesting rnAcr’s significant implications when considering new players in the host-bacteriophage evolutionary competition.

As primary producers, plankton play an important role in the marine environment as the basis of the food chain. Because of this, plankton biodiversity can be an indicator of the health of the ecosystem. As weather patterns change, rising salinity has been documented in marine environments across the globe. This has shown harmful effects in several organisms, including plankton. This study focuses on examining the correlation between salinity and plankton biodiversity across different seasons in Possession Sound located in Everett, Washington. For each site and season I compared biodiversity values with halocline salinity data for each plankton tow. Plankton were collected using horizontal plankton net tows at the halocline and identified based on morphology. I used the Shannon Diversity Index to examine biodiversity of phytoplankton and zooplankton. I collected this data on research cruises held by the Ocean Research College Academy and used historical data collected by previous students. The collected data encompass three sites visited from 2014-2024. Preliminary analysis suggests that biodiversity and salinity will have an inverse relationship. By examining the correlation between salinity and plankton biodiversity, we can begin to understand the potential impacts of rising salinity levels in marine ecosystems.

Escherichia coli (E. coli) abundance is commonly used to indicate water quality and environmental health. The pH of water has been shown to affect the survival of E. coli. Possession Sound is an estuary that faces a wide range of pH (around 7.5-9.0) throughout the year due to alkaline salt water from Puget Sound mixing with acidic fresh water from the Snohomish River. Primary production, organism respiration, nutrient runoff, carbon emissions, and currents also affect pH levels. This study aims to analyze the relationship between pH and E. coli abundance in an estuarine environment. PH and E. coli data was collected from 2018 to 2023 by myself and other Ocean Research College Academy students. PH data was collected with a YSI EXO2 Sonde. E. coli data was collected using a Niskin bottle to obtain water samples which were then transferred to Petri dishes for growing and counting E. coli. My preliminary analysis shows that Possession Sound’s average pH range is around 7.5-8.5, with pH being higher in spring and summer than in fall and winter. Early analysis using Spearman’s Rank Correlation suggests that pH and E. coli have a weak, inverse relationship. There is minimal research on the relationship between pH and E. coli in a marine setting, so my study helps to provide insight into the relationship between E. coli and pH in a unique estuary.

The properties of an estuary are driven largely by the vertical circulation and stratification patterns resulting from the convergence of dense salt water and less dense fresh water. These density-driven circulation patterns play an important role in the way vital nutrients are distributed throughout the environment to primary-producers. This study focuses on the seasonal variation in salinity and chlorophyll in comparison with river discharge levels of the Snohomish River, at Mount Baker Terminal, WA, within Possession Sound. This site is located 3.6 miles SW of the Snohomish River output near Mukilteo, WA. Salinity, chlorophyll, and depth data were collected from January 2020 to December 2023 using a boat based CTD (EXO SONDE). I participated in the collections from Fall 2020 to December 2023. I also reference the United States Geological Survey (USGS) streamflow data at site number 12150800. I categorized deployments by season (Winter: Dec-Feb; Spring: Mar-May; Summer: Jun-Aug; Fall: Sept-Nov) and utilized RStudio and GGPLOT2 to visualize and analyze my data. Preliminary analysis suggests well-defined haloclines and increased chlorophyll levels during spring months, corresponding with increases in river flow at this time. Decreased stratification, weak haloclines and low chlorophyll levels are conversely observed during summer months, corresponding with consistently low rates of river discharge. This raises questions concerning the impact that seasonal changes in river flow have on primary production at and around the halocline. Further analysis of chlorophyll ranges at the halocline is required to better understand this relationship. Monitoring the impact of river discharge variability and primary production is especially relevant in light of changes in seasonal weather patterns related to climate change.

Nutrients play a large role in marine primary productivity, and fluctuations in concentrations can have large impacts on the health of both marine life and human populations. Phosphates, silicates, nitrates, nitrites, and ammonium are all nutrients which are naturally present and key to the functioning of marine environments; however, negative consequences such as Harmful Algal Blooms (HABs) can occur if concentrations differ too far from normal ranges. Both natural processes and anthropogenic activities influence nutrient concentrations in marine environments, and in estuarine environments, like Possession Sound–part of the Whidbey Basin–the impacts of anthropogenic activities are further magnified due to unique estuarine hydrodynamics and the close proximity to dense human populations. For this study, I analyzed three sites within Possession Sound, looking at nutrient levels in comparison with dissolved oxygen (DO) and chlorophyll concentrations (as a proxy for primary productivity). I collected data during numerous research cruises and used this in tandem with the Ocean Research College Academy (ORCA) historical data set. Water samples were sent to the University of Washington Marine Chemistry Laboratory to measure nutrient concentrations, and chlorophyll and DO data were collected through the use of a conductivity, temperature, and depth (CTD) sensor. Preliminary results suggest a strong seasonal trend in nutrient values, with the lowest abundance occurring during the summer months. I hypothesize that further analysis will suggest an inverse relationship between primary productivity and nutrients. This study can potentially offer insight into the health of the Possession Sound ecosystem by assessing the extent to which dissolved oxygen, chlorophyll, and nutrient concentrations are within normal limits and following expected seasonal patterns.

 Primary productivity is the basis of energy production in marine ecosystems and is primarily dependent on nutrients and sunlight to occur, specifically the 400-700nm range of light that is used in photosynthesis, known as photosynthetically active radiation (PAR). In the Possession Sound estuary, nutrient input from the Snohomish River and seasonal daylight patterns lead to high productivity in spring, summer, and fall. These patterns provide an opportunity to investigate the influence of PAR against the influence of nutrients, which would allow for a greater understanding of the potential effects of PAR from artificial light at night (ALAN) in an estuarine system. We utilized existing and ongoing data from the paired deployments of a Seabird 19+ and a YSI EXO 2-Sonde to obtain water column profiles of PAR, chlorophyll, turbidity, and dissolved oxygen from July 2022 to April 2024 at Mt. Baker Terminal (MBT) in Mukilteo, and data from a moored MiniDOT PAR logger at MBT and moored EXO sensors at MBT and the Everett Marina from October 2023 to April 2024. The moored PAR logger and EXO sensors were set to collect data at 1 and 15 minute intervals, respectively. Additionally, we collected a concurrent total of 30 plankton samples at MBT and Everett Marina after sunset using a 1.7L Niskin bottle and a 20µm plankton filter. Preliminary analysis shows no measurable PAR at night, though Marina potentially has more productivity at night than MBT based on plankton data. Due to the small timeframe of our research, future explorations should expand the available analysis of temporal variation along with increasing the scope of sites throughout the Possession Sound and greater Salish Sea.

Solar energy is a promising form of renewable energy that will play a major role in reducing carbon emissions. Perovskite-based solar cells have attracted significant attention due to their high power conversion efficiency (PCE), which reached 26.1% this year, surpassing commercial silicon’s (23.3%). High PCE, low cost of materials, and ability to be solution processed make perovskite solar cells a prime candidate to replace silicon. However, efficiencies are still below the theoretical limit and these materials suffer from limited operational stability. To tackle these problems, scientists have focused on minimizing active layer and interfacial defects which act as barriers for charge extraction in a solar cell, lowering the device efficiencies. Defects also electronically dope the perovskite layer, changing the recombination kinetics in the sample. The goal of this project is to quantify how the electronic doping and defect concentration of the perovskite sample is affected by surface passivation treatements via fluence dependent photoluminescence (PL) and time resolved photoluminescence (TRPL) spectroscopy. By solving the kinetic equations at the basis of charge recombination, we can extract the rate constants that correspond to different charge recombination pathways. We pioneer a global fitting analysis to simultaneously fit TRPL and PL measurements for robust determination of these kinetic constants that are subsequently used to determine the doping density of films before and after passivation. We show that the electronic doping density is higher than previously reported in literature, and that this doping is reduced with a surface passivation treatment. We collaborate with the University of Arizona to correlate our measured electronic doping density to electrochemically measured defect densities on the same samples. This work will provide an implementable tool to quantitatively assess electronic doping and defect density values for various perovskite compositions which will be useful for optimizing future solar cell devices.

Vulcanized rubber, the main component of tires, is prized for its chemical durability and thermal stability. These properties, however, make disposal difficult and contribute to the increasing problem of polymer waste. To broaden the applications of end-of-life tires, we developed a method to chemically upcycle polybutadiene, a primary component of vulcanized rubber, via selenium-mediated allylic amination. We hypothesized that functionalizing the backbone of crosslinked polybutadiene with sulfonamide groups—without breaking their double bonds—would result in favorable thermal properties, creating a new life for the crosslinked polybutadiene. We used infrared spectroscopy and scanning electron microscopy with energy dispersive X-ray spectroscopy to confirm the aminated crosslinked polybutadiene’s molecular structure and differential scanning calorimetry and thermogravimetric analysis to measure its changes in thermal properties. Our research has future implications for the reduction of tire waste and reprocessing of other end-of-life crosslinked polymers.

To form Human Immunodeficiency Virus type 1 (HIV-1) virions, the HIV-1 Gag polyprotein multimerizes, traffics to the cell membrane, assembles into virions, and buds as viral particles. HIV uses the infected cell proteins to support viral assembly and export from the cell. Although some cellular proteins have been identified that participate in viral assembly and budding, the spatial and temporal "cellular proteome" is not known. Insights as to the order of proteins involved in facilitating assembly and budding provide information on viral infection and potential therapy targets. I am utilizing a split-APEX2-mediated proximity labeling method to identify which host factors interact with Gag during assembly. The basis of the method is splitting APEX2 into two segments encoded into separate Gag sequences so that the APEX2 segments rejoin for complete enzymatic function when Gag dimerizes and multimerizes. Once the APEX2 enzyme reconstitutes, we activate it to biotinylate cellular host proteins within ~20 nm of Gag during the processes of virion formation. I aim to deliver this system to primary human CD4+ T-cells and macrophages via two lentiviral vectors containing transgenes for either AP-Gag-P2A-EGFP or EX-Gag-P2A-mCherry. Cells express the dual fluorescence markers EGFP and mCherry if both Split-APEX2 domains are present, and we enrich these cells by cell sorting. APEX2 is restored and enzymatically active, allowing proximity-dependent biotinylation of Gag-host cell proteins for SILAC-based quantitative proteomic mapping during virion assembly. We are using this method to quantitatively map the Gag-host protein "interactome" in the infected cellular microenvironment in subtypes of human primary CD4+ T-cells and macrophages. Therefore, this research studies Gag interactions of potential HIV-1 host dependency factors in CD4+ T-cells and macrophages, the natural cell populations for HIV-1 infection. Previously, we conducted a similar study in HEK293Ts, and we anticipate elucidating a similar Gag-host cell protein analysis with this study.

The peptide neurotensin (NTS) has been known as a regulator of dopamine neuron activity and its system, which modulates numerous functions in the brain. Although ample research has now demonstrated that NTS in Ventral Tegmental Area (VTA) increases dopamine release in some regions, much remains unknown about the endogenous sources of NTS in the VTA and the impact of physiological NTS release. Recent NTS mapping data from the Soden lab demonstrated that there is a NTS projection from Periaqueductal Gray (PAG) to the VTA and also to hindbrain regions including the ventral medulla. This project investigates the effect of this interconnection between these three regions on the dopamine system. Utilizing advanced techniques in circuit mapping, mice will be injected in the VTA and the ventral medulla with fluorescent Retrobeads or a retrograde virus (rAAV2 anti mcherry or GFP), which are taken up by synaptic terminals and migrate up the axon retrogradely to label cell bodies, one color assigned for each region. Following euthanasia, mice brains will undergo immunohistochemistry such as histology and in-situ RNA staining. Then, data will be collected using imaging microscopes for results and further analysis. If NTS neurons in the PAG have green and red expressions, this will indicate that the same population of neurons send axons to both downstream regions, compared to PAG NTS neurons with only one color, indicating the presence of two separate neuron populations. Experiments on retrograde mapping of NTS inputs will contribute to building onto our current knowledge about VTA-PAG-ventral medulla circuit and effects on dopamine neurons following their interplay. In the end, our goal is to establish a novel understanding of endogenous NTS signaling mechanisms, mediation of complex reward processes, and treatment targets with experimental outcomes, giving rise to the development of therapeutic interventions towards addiction and related psychiatric disorders.

Marine heterotrophic bacteria play a pivotal role in microbial community dynamics. This study aims to understand interactions within the microbial community of the oligotrophic (nutrient-poor) equatorial Pacific, specifically investigating how heterotrophic bacteria respond to the growth of autotrophic picophytoplankton. This experiment attempts to provide a more faithful representation of in-situ conditions, overcoming previous difficulties in capturing the dynamic behavior of microbial communities in the field. A novel methodology utilizing continuous chemostats with natural communities in the field accomplishes this objective. Three growth chambers were employed, two as chemostat systems and one in batch culture mode. All three growth chambers contained the natural microbial community that passed through a 3 µm pore-size filter. Chemostat systems continuously received 0.2 µm-filtered seawater (without microbes) while an equivalent volume was removed from the growth chamber simultaneously. Dissolved inorganic nutrients required by autotrophs – silicate, phosphate, and nitrate – were added to one of the chemostat’s 0.2 µm filtered media reservoir. This methodology was compared to a traditional batch culture, where nutrients were added to the growth chamber once, at time-zero. Cultures followed a 16-hour on/8-hour off light/dark cycle using LEDs, simulating the equatorial Pacific day/night cycle. Community responses were measured by continuous optical density measurements (OD), with endpoint subsamples analyzed for microbial abundance and DNA content using flow cytometry. Distinct day/night responses were observed in all cases, with the nutrient-enriched chemostat showing the most pronounced response. Overall, the results provide new insight into the linkages between marine autotrophic and heterotrophic microbes, while demonstrating an effective new methodology for examining microbial community responses to added nutrients. Thus, this study not only advances our understanding of microbial community dynamics in the oligotrophic equatorial Pacific but also introduces a novel experimental method that can be applied across a diversity of marine and aquatic environments.

Saltwater estuaries can experience high turbidity levels due to river input and tidal influences. Turbidity is a measure of inorganic and organic particles suspended in the water column. Reduced light penetration due to higher turbidity levels can contribute to decreased levels of primary production and the introduction of harmful pathogens to the environment. Understanding the relationship between river discharge, tides and turbidity levels could lead to a better understanding of the causes of turbidity in estuaries such as Possession Sound, WA. I hypothesize that higher current velocity contributes to higher turbidity levels. I analyzed data from a moored Acoustic Doppler Current Profiler (ADCP) and a Conductivity, Temperature, Depth (CTD) sensor located in the Everett Marina. ADCP and turbidity data were collected every 15 minutes, 24 hours a day, from 2017 to 2021. Preliminary results suggest that higher current velocity correlates to higher turbidity levels. Future research looks to discover how river discharge, tides and seasonal variance play into turbidity spikes.

The paradigm for manganese (Mn) cycling in the marine environment has shifted over the past two decades to include not only the +IV and +II oxidation states. It is now recognized that dissolved Mn(III) can also exist when stabilized by organic ligands. Mn is critical for sustaining life and influences the cycling of many other bioactive elements. Because of this, further research is needed for understanding how Mn cycles in the environment, both between physical and chemical phases. My project aimed to look at how Mn cycles between dissolved and particulate phases and its three environmentally relevant oxidation states along the salinity gradient of the Mississippi River delta and what role organic ligands play in mediating Mn transformations. I hypothesized that the salinity gradient would influence the availability of organic ligands, which would promote the oxidation of dissolved Mn(II) and particulate Mn oxides (MnOx), making the cycling of Mn during estuarine mixing more complex than previously understood. To test this, I collected water from the Mississippi River and the Gulf of Mexico to conduct a mixing experiment to model the salinity gradient. UV-Vis spectrophotometry was used to analyze particulate and dissolved Mn speciation as well as the characteristics of the organic matter present. Inductively coupled plasma-mass spectrometry was used in analyzing dissolved Mn and Mn flocculants. Preliminary results show increases of dissolved Mn during mixing, and potential loss of particulate MnOx. Combined, these suggest redox cycling of Mn during estuarine mixing impacts its solubility and ultimately transport to the Gulf of Mexico. This region experiences heavy nutrient loading that leads to seasonal hypoxia. Understanding the cycling and solubility of Mn is imperative because it has broader implications for redox processes and element cycling in the Northern Gulf of Mexico, especially during hypoxic events.

Over the last two decades, the threat of climate change has inspired significant research in the Salish Sea. Understanding trends and correlations between water temperature, dissolved oxygen (DO), and chlorophyll levels can help us understand how climate change and other anthropogenic activity has already affected the Salish Sea. My reseach focuses on seasonal and annual trends of water temperature, DO, and chlorophyll levels between 2019 and 2023 in Possession Sound, located in Everett, Washington. This longterm data-stream is generated by the Ocean Research College Academy, collected autonomously every 15 minutes by a pair of EXO sondes that are moored at Mount Baker Terminal and Everett Marina. My goal is to understand the relationship between water temperature, DO, and chlorophyll seasonally and historical trends over multiple years in Possession Sound. Preliminary figures and outside research have shown fairly consistent seasonal cycles for temperature and chlorophyll. DO trends are not as clear and data suggest significant variation is occurring within a short time frame. Future reseach may include comparing river discharge data to water chemistry data, however a more comprehensive understanding of specific inputs to the Snohomish River system is needed to draw solid conclusions about the affects of climate change.

A critical feature of autonomous cars is the ability to follow a road or predefined path. Classical methods often rely on extensive prior mapping with precise GPS positioning. These methods are labor intensive and struggle with changing, unstructured environments. Instead, machine learning (ML) models are trained to recognize paths and follow directions. In this work, we combine simulated and real-world data to train a neural network policy that controls an autonomous ground vehicle down a hallway, avoiding collisions. Training a ML road-following model consists of three steps: data collection and preprocessing, model training, and model evaluation. While all three steps pose challenges, collecting high-quality, real-world data can be expensive and dangerous in road environments. Because of this, simulator data is useful as it allows for data to be collected safely and inexpensively. Thus, we study how much the required amount of real-world data can be reduced to successfully train a road-following robot with the use of simulator data. So, we collected simulator data using AirSim to train a convolutional neural network that follows a path in simulation through live environment images. We then fine-tuned the model using real-world data collected from MuSHR cars through hallways of a building. Next, we test the fine-tuned model on the simulator to ensure limited degradation to the model solely trained from AirSim data. Finally, we deploy the model on a robotic car in a real-world environment and evaluate the model’s performance compared to the baseline model trained on real-world data. We demonstrate that we can successfully train a model in simulation (MSE <= 0.01radians), and we expect to show a comparable performance in reducing the number of collisions and minimizing trajectory differences between expert and learned controller from a model trained on simulator + less real-world data and a model trained solely on real-world data.

Heart disease is the leading cause of death in the United States. Due to the inability of cardiac tissue to self-heal, extreme cases necessitate heart transplants and most patients do not fully recover. A promising novel approach to create engineered heart tissue focuses on 3D extrusion-based bioprinting of stem cell-derived cardiomyocytes. Mature iPSC derived cardiomyocytes, the cells responsible for the contraction of the heart, do not proliferate. Therefore, the printed tissue construct must be created with the final desired cell-density de novo in order to mimic native cardiac tissue. Researchers need a viable method for extruding high cell-density bioinks to form functional constructs. In my research project, I am working  to generate 3D bioprinted cardiac tissues with high cell-density to measure both electrophysiological characteristics and contractile force output. The use of cell-only bioinks is atypical, and there is limited research in the literature documenting its properties. I measured the acute change in viability of NIH 3T3 cells extruded through a needle to investigate the effect of the needle’s hydrodynamic forces on the cells at high density. The data shows that cells extruded at high density maintain a high viability, and we observed strong structural cohesion in the extruded filaments. I optimized the extrusion parameters, needle diameter and flow rate, to create long-lasting filaments, and constructs remained intact over a 5 day observation period. However, we found they fail easily with agitation. Further work is needed to optimize bioink and conduct further studies using flexible posts to measure contractile force output and calcium imaging to determine the electrophysiological characteristics of our cardiac constructs. Quantifying these properties is critical to ensuring that constructs recapitulate the characteristics of native cardiac tissue. This research may aid in the development of engineered cardiac tissue for transplantation and drug discovery.

Repeat thrombotic events have been associated with increased levels of Von Willebrand Factor (VWF) in patients prescribed dual antiplatelet therapies (DAPT), medications designed to prevent thrombosis. VWF is a protein that regulates platelet adhesion during hemostasis, allowing platelets to aggregate at sites of vascular injury. A microfluidic device containing a rigid block that simulates vascular injury and a flexible post to measure platelet contractile force through its displacement will be used to form shear-induced thrombi. I will determine how VWF affects platelet activation by testing whole blood samples and samples doped with DAPT (0.3 mM ASA and 10 μΜ 2-MeSAMP), 50 μg/ML VWF, or both VWF and DAPT. Platelet activation is measured through the area of platelet-plug, intracellular calcium levels, and platelet-plug contractile force. Preliminary experiments have shown that high VWF levels produced the largest platelet plugs whereas adding DAPT led to opposite effects. My results present that adding both VWF and DAPT to whole blood leads to similar platelet activation and larger platelet plug size as whole blood doped solely with VWF. The data obtained from this research can provide new insights into the improvement of therapeutic agents that aim to target VWF’s interaction with platelets and ultimately prevent repeat thrombotic events.

Heart disease takes an estimated 17.9 million lives each year, highlighting the pressing demand for cost-effective treatments. Melusin, a chaperone protein in the heart, holds potential as a target for heart failure therapeutics. Previous studies done in wild-type (WT) and melusin knockout (MelKO) mice discovered the absence of melusin was associated with a hypertrophic response indicative of heart failure. I plan to investigate the biomechanical role of melusin in humans using human-engineered heart tissues (EHTs) created from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) that lack melusin and their isogenic controls. EHTs are a 3D in vitro model of the human heart, ideal for studying the role of melusin in humans. I hypothesize that WT EHTs subjected to mechanical stress will outperform the MelKO EHTs. The EHTs are suspended between one flexible and one rigid silicone post. The EHT displaces the flexible post as it contracts, from which the displacement can be measured to calculate various auxotonic properties of the tissue. To induce mechanical stress on the tissues, I use a brace to restrict the movement of the flexible post. I am using histology to determine if there are any morphological differences between tissue types resulting from the brace. Thus far, I have cast WT and MelKO EHTs and completed twitch force measurements two and three weeks post-casting. Overall, I found the MelKO EHTs demonstrated lower contractile force than the WT EHTs. I plan to cast and collect more EHT data with and without braces in order to provide insight into the role of melusin in humans. Furthering our understanding of the heart’s mechanotransduction properties using EHTs is important in expanding our knowledge about the various pathologies of the heart. Ultimately, studying the pressure overload pathways involving melusin can lead to the development of future therapies for cardiovascular disease.

Current technology to control 3D cell function takes advantage of bioorthogonal photochemistry to immobilize proteins into materials by using photocages—photoremovable molecular groups that block protein activity. However, diffusion limitations necessitate patterning times ranging from hours to weeks, far longer than the timescales of many biological processes. Protein activation aids signaling events within the extracellular matrix (ECM), leading to downstream changes in cell fate and physiological responses in our bodies. In order to probe and investigate biological systems, hydrogel materials provide an ideal synthetic platform, due to their polymeric, water-swollen characteristics that mimic the native ECM. In this project, I will use light to control the spatial and temporal presentation of biochemical cues through the photoactivation of proteins within hydrogels. We hypothesize that the kinetics of the protein activity between solution and biomaterial studies should correlate, given their dose-dependent response to light exposure—by varying the intensities of light and time intervals of exposure. By characterizing the photoactivatable protein system and controlling protein activity, we intend to use this platform to photoactivate biologically relevant proteins to control signaling that occurs on shorter time scales, applicable to biochemical processes.

 Injectable biomaterials can be used for targeted, local delivery of drugs and small protein factors. However, designing a biomaterial that is self-healing, bioresorbable, highly-defined, and allows for the independent release of multiple protein drugs remains a challenge. Interpenetrating network (IPN) hydrogels formed by physical crosslinks provide a solution as they combine the properties of two networks into one material, as well as form gels that are injectable and self-healing. Our project aims to produce the first ever reported fully recombinant protein-based IPN hydrogel that can be used to deliver multiple drugs simultaneously. Recombinant protein-based polymers are advantageous for forming biomaterials due to their bioactivity, biocompatibility, and tunability. Each biopolymer contains coiled-coil proteins linked by a flexible hydrophilic linker that is bisected by a click-like chemistry protein system, known as a polyproteam. These polyproteams allow drugs or protein factors to be covalently ligated to the networks, so that drug delivery is dependent on material erosion instead of diffusion. We characterized the material’s properties using rheometry to measure material stiffness, percent strain at which the material liquifies, and self-healing behavior. Then, we tested the ability of our hydrogel to deliver drugs by ligating epidermal growth factor and insulin growth factor to the hydrogel and measured the release of protein over time through a Western Blot. The bioactivity of the growth factors was confirmed by culturing with human mesenchymal stem cells (hMSCs) and using a Western Blot to measure activation of the ERK signaling pathway. If successful, the IPN hydrogel can be used for clinical applications, as a tool for drug delivery.

Gas-phase emissions from sea-spray generate aerosols which are an important source of atmospheric halogens. Halogens (chlorine, bromine, and iodine-containing species) are important in the atmosphere because they affect the abundance of greenhouse gasses such as ozone and methane. The Bermuda boundary Layer Experiment on the Atmospheric Chemistry of Halogens (BLEACH) is a campaign that studies the abundance and cycling of atmospheric halogens. Filter samples from field campaigns are often frozen to preserve them for future analysis. However, after freezing a mixture of anion standards that replicate atmospheric composition for measurement on an Inductively Coupled Plasma Mass Spectrometer (ICP-MS), total aerosol iodine showed a tenfold increase in concentration in two separate trials compared to room temperature. Understanding the impact of freezing filter samples on aerosol iodine is crucial in interpreting BLEACH observations and could change the understanding of aerosol iodine speciation in the scientific community. I investigated this total iodine enrichment after a series of experiments on frozen and room temperature laboratory standards using Ion Chromatography (IC), which measures iodate and iodine separately. The tenfold iodine enrichment observed after freezing measured on ICP-MS was not replicated in IC trials. The total iodine ratio of frozen to room temperature was 1.1 on the IC and 9.8 on ICP-MS. Our results also show that the ratios iodide/iodate are the same for frozen (1.3) and room-temp (1.3) samples, suggesting that the conversion between iodide and iodate is not responsible for the enrichment in ICP-MS. Our observations of total aerosol iodine concentrations in Bermuda’s atmosphere are consistent with previous studies in the same region. This either suggests that the iodine enrichment after freezing is unique to the “simulated atmosphere” standard prepared in this study, or all field observations using ICP-MS may be overestimated by an order of magnitude.

In a recent breakthrough, bullvalene, renowned for its “shape-shifting” molecular nature with over 1.2 million degenerate isomers, has been successfully integrated into polymer backbones. This integration addresses challenges in solubility and thermal properties crucial for tailoring polymers used in manufacturing diverse products ranging from phone screens to organic solar cells. This project aims to deepen our understanding of the interplay between fluxionality and thermal properties by examining the thermal stability of small molecule bullvalene models. Through extrapolating insights for bullvalene-substituted polymers, our research seeks to contribute to the advancement of the development of advanced materials suited for varying thermal conditions. We synthesized small molecule bullvalenes to mimic polymer chains, subjecting them to diimide reduction to suppress fluxionality before comparison with their fluxional counterparts. Their thermal properties were characterized using Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC). Key findings reveal a decrease in glass transition temperature upon reduction of bullvalene, highlighting the impact of fluxionality on thermal stability. Future work will delve deeper into exploring the thermal properties of small molecule models, providing insights into polymer behavior. We anticipate bullvalene as an internal plasticizer capable of modulating rigidity, solubility, and thermal properties within different classes of polymers, thus enabling a more efficient and cost-effective large-scale industrial production of a wide array of polymeric materials.

Corepressors are proteins found in all eukaryotes that work with DNA-binding proteins to repress many genes. Keeping some genes off, yet ready to quickly turn of if needed, is essential for development and physiology. Our project aims to identify interacting proteins that work with TPL, a conserved plant corepressor, to form a transcriptional repressor complex. To uncover these proteins, we created a visually screenable plant line containing RUBY, a reporter that expresses throughout the plant, turning it dark pink to purple. We next created a synthetic repressor dCas9-TPL and guide RNA (gRNA) construct that binds to and represses the RUBY reporter. Roots of plants with both constructs appeared whitish-pink, indicating dCas9-TPL is transcriptionally repressing RUBY. We then mutagenized 40,000 individuals from this line using the chemical Ethyl methanesulfonante (EMS), which creates new point mutations in random locations throughout the genome. We identified 257 individuals from 129 mutagenized families with dark pink roots, which show that repression by dCas9-TPL has been impaired. Many of these adult plants had phenotypes in addition to appearing pink, including miniaturization, infertility, and irregular growth patterns, suggesting that the mutations we found are affecting other pathways that require TPL. Using Mendelian genetics, we are currently characterizing the mutation types (i.e. homozygosity, recessive, or dominant) as well as establishing complementation groups. We will then backcross the lines with the parent line to eliminate extraneous mutations and perform whole genome sequencing to determine the precise mutation causing loss of repression. This will also tell us if repression was due to mutating a TPL interactor, or mutating one of our reporter or repressor constructs. By finding genes required for TPL to act as a corepressor, we hope to understand conserved mechanisms of corepressor activity across diverse eukaryotes.

In response to changing conditions, organisms express genes to optimize the match between their phenotype and the environment. Understanding the mechanisms for how genes are turned on or off is therefore an important research area. One challenge in conducting this research is that many of the proteins involved in regulating gene expression are essential to life, and disrupting their function can lead to death. My research focuses on the essential gene SPT6, which encodes a protein that works with RNA polymerase during the elongation phase of transcription. Recently, the Nemhauser Lab has found that SPT6 also plays a role in transcriptional repression. My project aims to differentiate the role that SPT6 plays in transcriptional activation and repression by disrupting its expression in Arabidopsis. Given that SPT6 mutants do not survive, here I test the use of a new tool that allows me to remove my gene of interest in a particular tissue at a particular time. The tool is based on a molecular switch that relies on serine integrases which can recombine DNA between two specific sequences. So far, I have worked with my mentor to rescue SPT6 mutants with a target that expresses the wild-type version of SPT6. Once the integrase is expressed, the recombination turns off the SPT6 gene and turns on a fluorescent reporter. I express the integrase from a promoter that is active only in the first stages of making a new root, so I can observe the impact of loss of SPT6 function in a cell type unnecessary for plants to survive in lab conditions. This project promotes an understanding of the multiple roles of SPT6 during the transtition from repression to activation, and as SPT6 is highly conserved across eukaryotes, my work in plants may also contribute to understanding human diseases. 

Some genes are on all of the time in most cells, and carry out functions that are essential for life. Unsuprisingly, essential genes are difficult to study, as interfering with their function leads to death. One such critical component is the multi-protein Mediator complex, which is found at every eukaryotic promoter where it coordinates activation of gene expression. My project focuses on one of the core components of the Mediator complex, MEDIATOR21 (MED21). While MED21 is required for gene activation, the Nemhauser Lab recently found that it also plays a role in repression of gene expression through interaction with the corepressor protein TPL. I would like to be able to differentiate the role MED21 plays in activation versus repression using the plant model Arabadopsis. This work is made more complicated by the fact that most mutations in MED21 lead to lethal phenotypes. As an alternative I recently developed a new technology called a molecular switch that turns off MED21 in certain tissues or in reponse to addition of a chemical. The molecular switch relies on the expression of serine integrases that recognize, and recombine the DNA between, two specific DNA sequences. By expressing an integrase portein from a promoter that is only expressed in secondary roots, I can study MED21 loss of function in a small pool of stem cells while the rest of the plant is wild type and healthy. Plants that have undergone this cell-type-specfic switch exhibit several abnormal root phenotypes including agravitropism, increased root formation, and more root hairs. My next experiments include uisng a switch from wild-type MED21 to a mutant form incapable of binding to the corepressor TPL. This study will help us better understand the role MED21 plays in repression versus activation, and how state switching contributes to organogenesis.

The COVID-19 pandemic heavily affected individuals’ lives, not only through disease transmission but downstream effects such as unemployment and worsening mental health. People living with HIV (PLWH)—as a marginalized and vulnerable population—experience greater mental health risk and life challenges than the general population, which the COVID-19 pandemic exacerbated. Studies that investigate PLWH’s wellbeing during the pandemic are few. In this study, I conducted a mixed-methods study in Qingdao, China using interviews and surveys to explore the effects of COVID-19 and associated policies on the wellbeing of PLWH. My overarching aim was to understand personal experiences of wellbeing, mental health, and factors specific to PLWH before, during, and after the COVID-19 pandemic. Collaborating with Qingtong, an NGO working with PLWH in Qingdao, China, I conducted 15-minute semi-structured interviews with a total of 17 HIV+ individuals whose median time had been living with HIV was 6 years (+/- 2.32). I asked questions about life and wellbeing changes due to the pandemic and challenges they faced. I qualitatively coded the interview transcripts, and found three themes, including (1) Social embodiment, support, and stigma of living with HIV, (2) COVID’s physical and mental effects on PLWH, and (3) Medication access changes over the pandemic. Overall, I suggest that mental health issues experienced by PLWH were not caused by their HIV status solely but by systemic factors such as COVID-19 policies and geographic disparities in accessing medication. Therefore, I advocated that the management of PLWH and relative policies need to be further strengthened in order to respond to any future public health emergencies and to ensure the wellbeing of this population.

Upconversion (UC) is a non-linear optical process where a material absorbs two lower energy photons and subsequently emits one of higher energy. Currently, inorganic UC materials used in lasers and photovoltaics are primarily lanthanide-based. However, a few transition metals also exhibit UC, such as Re4+ , Os4+, Ti2+, Ni2+, and Mo3+, and due to their high oscillator strengths, d-d transitions, and a strong ligand field dependency, offer the potential for greater tunability and efficiency in upconverting optoelectronics than their than their lanthanide counterparts. The goal of this work is to increase Re4+’s PLQY by isovalently doping low-phonon vacancy-ordered double perovskites (A2BX6 : A = Cs+, NH4+; B = Ti4+, Zr4+; X = Cl-, Br-) with rhenium to minimize non-radiative decay that can occur through defects and lattice vibrations. This has been attempted via schlenck line synthesis of the host lattice and coprecipation and ion-exchange doping procedures. While [ReX6]2- has previously demonstrated near-IR to visible upconversion in the bulk, this work aims to characterize its upconversion mechanism on the nanoscale with variable temperature and time-resolved photoluminescence. If made successfully, the colloidal stability of Re4+:Cs2TiBr6 nanocrystals would allow for new post-synthetic processing avenues including electrohydrodynamic inkjet printing and core-shelling, and new applications in flexible electronics.

Black phosphorus (bP), an allotrope of phosphorus, is a 2D Van der Waals material composed of corrugated layers of phosphorus atoms. Few-layer bP is a semiconductor with interesting physical properties, including relatively high carrier mobility and layer-dependent band gap. These properties may be harnessed for applications including nitrogen fixation photocatalysts, thin film transistors, and sensing devices. One limitation that must be overcome before bP can be used in devices is its degradation into phosphoric acid when exposed to oxygen, water, and/or light. Finding passivation methods is crucial for the future use of bP in electronics or photochemistry. As each passivation treatment changes bP’s electronic properties, it is important to find protection methods that are compatible with each use case. To investigate possible methods to reduce surface degradation, we exfoliate bP in solution and treat it with a passivation candidate. We then use ultraviolet-visible light (UV-Vis) spectroscopy to track the amount of unoxidized bP that remains in solution during ambient exposure. Since bP absorbs strongly across the UV-vis region, while the decomposition products, phosphorus oxides, do not, UV-vis is an ideal method for measuring degradation. Possible treatments include attaching alkoxy or thiolate groups via peroxides or disulfides to bP edges to protect the particularly reactive dangling bonds, treatment with radical scavengers such as butylated hydroxytoluene, or noncovalent protection with Tetracyanoquinodimethane (TCNQ).

Science is rapidly evolving, yet its advances do not enter classrooms at the same rate. Systems Education Experiences (SEE) is a program in the Baliga Lab at the Institute for Systems Biology (ISB) that accelerates the transfer of scientific knowledge and practices to classrooms. One active area of Baliga Lab research is elucidating the level of resilience organisms have, when faced with complex environmental changes. My role is to design laboratory experiments that investigate this with the model organism Halobacterium salinarum (Halo) and to connect this to broader rules governing natural systems for use in high school classrooms. My first experiment probes the resiliency of Halo with the introduction of a combination of stressors (salt and hydrogen peroxide) and its recovery after population collapse. The second measures the long term phenotypic changes in the population. I wanted to see if after being exposed to a new environment if there was an advantage to having gas vesicles and if it is an irreversible trait that allows Halo to be resilient across a variety of environmental conditions. This relates to broadly applicable rules governing resilience across many systems. This project serves as a model for how all organisms respond to stress. Combinations of stressors in human lives can make us less resilient. However, strategies to quickly prepare, respond, and react can improve outcomes for individuals and the overall population. This project connects to a goal of K-12 science education which is to not just teach academic concepts but to equip students with knowledge that can be applied to all parts of life. Our knowledge on the mechanisms that control how organisms respond to stress is extremely limited. By understanding the biological stress response we can promote resilience in the earth's most vulnerable systems in the wake of climate change.

The aorta is responsible for transporting blood to all of our organs and extremities. During an aortic dissection, a tear develops in the intima (inner) and media (middle) layers of the aorta, causing a rapid influx of blood and a consequent separation of the adjacent layers. Type A aortic dissections (TAAD) refer to aortic dissections proximal to the left subclavian artery – the aortic segment nearest to the heart. The mortality rate of TAAD is shockingly high: roughly 50% for both acute and long-term survival. There are numerous factors contributing to long-term survival, and current literature has primarily focused on medical/surgical interventions to prevent disease progression. There is comparatively little data regarding epidemiology and social determinants that are relevant to long-term survival. This project aims to analyze the impact that socioeconomic factors, gender, and race have on short-term and long-term surgical outcomes of TAAD patients. A TAAD surgery database at the UW Medical Center is the primary point of investigation. In this study, I am assisting with the primary analytical design and statistical analysis of our data through the use of the R programming language, which will be done in conjunction with a biostatistician. I am also responsible for helping synthesize and communicate our findings in the form of an abstract and manuscript. Preliminarily, we have begun to see correlations between the aforementioned population characteristics and short-term surgical outcomes. As we continue with more complex statistical analyses, especially regarding long-term survival, we expect to see noticeable differences between patients of different socioeconomic, gender, and/or ethnic groups. Our project has the potential to benefit all TAAD patients. A better understanding of health disparities among these patients will allow for the identification of modifiable risk factors and targeted use of resources, thus improving care and outcomes in all associated populations.

Located in the Southern Pacific Ocean, American Samoa was formed nearly 400 thousand years ago due to hotspot volcanism. As these eruptions occur, ash and volcanic rock fragments settle and leave behind texture, roughness, and clast sizes that are identifiable using mapping techniques such as backscatter analysis. The 2024 Oceanography Senior Thesis cruise aboard the R/V Thomas G. Thompson, produced bathymetric and backscatter maps utilizing the Multibeam Kongsberg EA302 to identify the boundary of such deposits and the thickness of sediment that has been deposited on it, indicating relative age and formation of volcanic features on the seafloor. This study focused on the islands Ofu-Olosega and Ta’u, and we located several intact and exploded cinder cone. Sediment cores were collected to quantify the grain size of the basalt that erupted violently out of these hot spots. We hypothesized that the grain size would correlate with distance from the caldera, with larger clasts sinking closer to the eruption site, and fine sediment carried farther. This was found true, but there were also large grain sizes radiating away from the initial cinder cone site, indicating the presence of other eruptions on the seabed. Multiple landslides were documented on the southern and northeastern slopes of Olosega Island. These landslides display key features such as steep amphitheater headwalls, blocky ridges, and hummock aprons. The landslides were classified as either slumps or debris avalanches based on these characteristics and compared to other volcanic hotspot landslides within the Pacific region. We hypothesized failure deposits would be identifiable in the seabed up to 30 km away from the caldera, and found them to be graphically obvious for about 21 km.

Bioconjugation, or the covalent linkage between a biomolecule and another chemical group, creates hybrid "conjugates" that exhibit the properties of both biomolecules and exogenous moieties. The N-termini of proteins often fall outside of their final fold, making the N-terminus an optimal site for conjugation while preserving a protein’s native folding and bioactivity. Consequently, N-terminal modification of proteins and peptides has been a long-standing goal in fields like drug delivery, biotherapeutics, and cellular imaging. However, the current techniques for N-terminal protein conjugation are limited by either the introduction of bulky protein assemblies at the conjugation site, the need for multiple costly and complicated steps, or low site selectivity. In this project, we aimed to develop an improved route for N-terminal bioconjugation. We created a generalizable platform for single-step purification and near-scarless N-terminal bioconjugation of proteins by leveraging the chemistry of the atypically split intein VidaL. To evaluate the effectiveness of our platform, we first examined the kinetics and reaction conditions of VidaL bioconjugation, confirming its ability to modify the N-termini of proteins successfully and selectively. Then, we used our platform to conjugate an alkyne, biotin, or FAM-biotin moiety to the N-termini of fluorescent proteins (EGFP and mCherry), a model enzyme (beta-lactamase), and a model growth factor (EGF). Through measuring fluorescence and conducting nitrocefin and proliferation assays, I found that, regardless of the moiety added, bioconjugation did not impact the native function or activity of these proteins. In the future, we expect that this platform's ability to easily N-terminally bioconjugate proteins with minimal impact on their functionality will find use across the growing fields of applied chemical biology.

Understanding the complex neural mechanisms underlying motivated behavior and reward processing is crucial for advancing our knowledge of addiction and related disorders. In this study, we explore the role of neurotensin (NTS) peptides in modulating dopamine neurons within the ventral tegmental area (VTA), a key brain region implicated in reward processing. Previous research has highlighted the reinforcing effects of optogenetic stimulation of NTS inputs from the lateral hypothalamus to the VTA. However, a gap remains in understanding how NTS signaling interacts with other brain regions involved in motivated behavior. Our research addresses this gap by investigating the contribution of NTS and fast neurotransmitters in circuit-specific behavioral activation within the VTA. We hypothesize that distinct NTS inputs differentially influence motivated behavior in a combined NTS and GABA/glutamate-dependent manner through optogenetics and circuit mapping. We employ a multidisciplinary approach combining optogenetics, CRISPR gene editing, and behavioral assays to achieve this. Specifically, I utilize optogenetic stimulation to selectively activate NTS inputs from diverse brain regions, including the periaqueductal gray (PAG) and the pedunculopontine tegmentum (PPTg), targeting specific input regions via unilateral injections of AAV1-FLEX-Chrimson-mCherry. Control mice receive AAV1-FLEX-mCherry alone to ensure the specificity of our manipulations. Following surgical procedures and acclimation to the experimental setup, I conduct behavioral assays on mice, specifically Real-Time Place Preference (RTPP) and open-field testing to assess the effects of optogenetic stimulation on locomotion and anxiety-related behaviors. Subsequently, we investigate the mediating role of NTS and neurotransmitters by employing CRISPR viruses to target genes related to different components of neurotransmission. By integrating cutting-edge techniques with behavioral assessments, our study aims to elucidate the intricate interplay between NTS signaling, fast neurotransmitters, and specific circuit activations within the VTA. These findings contribute to our understanding of reward processing and hold potential implications for developing novel addiction treatment strategies.

Biological processes depend on the intricate functions of proteins, which serve as the driving force in vital reactions. Given the importance of proteins in biological processes, several methods have been developed to control their functions in biomaterial and in vitro. Current methods often rely on secondary fixatives and caustic chemicals, posing risks of functional impairment in target proteins. To address these limitations, my project focuses on modifying cells with non-endogenous machinery, enabling expression of photoactivatable proteins – those with photocaged amino acids installed at essential locations, and using them to optically stimulate protein function. This strategy enables the triggered activation of target proteins with light, mitigating the risks associated with chemical interventions. We predict that the degree of protein activation can be controlled in a dose-dependent manner by manipulating light exposure duration and intensity. Genetic code expansion is used to integrate noncanonical amino acids, site specifically, into proteins expressed in bacteria and mammalian cells. By incorporating these photoactivatable proteins into biomaterials and living cells, our research aims to gain insights into fundamental cell signaling processes through control of targeted proteins. By analyzing biological mechanisms at the molecular level, we can guide cell processes and make informed decisions regarding cellular behavior and fates.

Prostate cancer is the most common type of cancer amongst men in the U.S. It relies on androgens that bind to the androgen receptor (AR), which increases the transcription of genes associated with the growth and proliferation of the prostate cells. For the AR-driven prostate cancer (ARPC), current treatments involve decreasing androgen levels (Androgen Deprivation Therapy) or inhibiting the ARPI (Androgen Receptor Pathway Inhibitors). However, around 15% of patients develop resistance to these treatments, resulting in a type of prostate cancer called neuroendocrine prostate cancer (NEPC). NEPC cells are no longer dependent on AR activity, which makes this subtype difficult to treat with the current treatment options in the clinic. To better understand the biology of NEPC, we focused on gene expression at the protein synthesis level and found that NEPC has a decreased level of a tRNA called Arg-TCT-1-1. Following Arg-TCT-1-1 tRNA overexpression in NEPC, we detected elevated expression of AR downstream targets via qPCR and western blot. NEPC with high Arg-TCT-1-1 also responded to an AR inhibitor called enzalutamide as measured by cell viability assays. To further investigate the role of Arg-TCT-1-1 in prostate cancer, we used shRNA-mediated knockdown of this tRNA in prostate cancer cells with high AR expression and measured changes in gene expression. This study will provide important insights on the role of Arg-TCT-1-1 during the differentiation process from ARPC to NEPC.

The aim of this study was to analyze the three-dimensional deformational changes in the tongue base during natural chewing in a minipig model. Eight 7-8-month-old Yucatan minipigs were used in this study. Under anesthesia, eight 2mm ultrasonic piezoelectric crystals were implanted in the tongue base forming a cubic-shaped configuration, representing the right/left dorsal (RDL-LDL) and ventral (RVL-LVL) lengths, anterior/posterior dorsal (ADW-PDW) and ventral (AVW-PVW) widths, right/left anterior (RAT-LAT) and posterior (RPT-LPT) thicknesses. After the minipig was awakened after anesthesia withdrawal, unrestrained feeding was offered for 10-15 minutes. The amplitudes and onsets for each dimensional change of the crystal-circumscribed region were measured from the start of the jaw opening phase of chewing, and up to 21 concessive chewing cycles were measured. The phases of jaw opening-closing/power stroke during chewing were determined from the simultaneous electromyographic recordings. The duration measurements represented the chewing cycle lengths. The ADW was set up as the reference dimension for the onset calculation (zero point) due to its most stable nature during chewing. All measured dimensions showed either increased (peak/elongation) or decreased (valley/shortening) signals from the baseline. Overall, lengths (RDL-LDL and RVL-LVL) are either shortened or elongated depending upon a given chewing side. The widths (ADW-AVW and PDW-PVW) increased, and the RPT-LAT thickness increased while RAT-LPT thickness reduced during jaw opening of chewing. RDL showed the largest shortening (-42.28% valley-amplitude) while LDL showed the largest elongation (21.15% peak-amplitude, p ≤ 0.05). Earlier onsets occurred in shortening and later onsets in elongation (1.41% to 10.53%) in relation to the reference. Last, the duration of the chewing cycle was 0.45-0.58 seconds. The findings of this study showed a specific kinematic pattern of the tongue base in chewing. This will contribute to a better understanding of the biomechanics of the oropharyngeal function.

With the increasing use of lithium-ion batteries, the trajectory of the modern world’s energy needs calls for an improvement in their safety and functionality. Current lithium-ion batteries use a lithium salt dissolved in organic carbonates, which results in a liquid electrolyte with high lithium dissociation and fast conductivity. However, the use of organic solvents makes these electrolytes flammable and prone to combustion in the event of a dendritic formation causing a short. One approach to potentially improving these electrolytes is a solid single-ion polymer electrolyte where the anion is part of the polymer chain to potentially allow higher conductivity and lithium transference numbers. Our approach is to synthesize single-ion copolymers with varying weight percentages of a single-ion monomer (SIM) to control the ion concentration and a monomer with an oligomeric ethylene oxide sidechain (ONDI-12) to lower the glass transition temperature. The goal of my work is to identify the ratio of the two monomers that gives optimal conductivity, improving the potential use of our copolymer as a solid single-ion copolymer electrolyte in lithium-ion batteries. Initial electrochemical impedance spectroscopy measurements of our previous copolymers indicate that lower ion concentrations and lower glass transition temperatures resulted in increased conductivity with 20 wt.% SIM. Building from this work, I synthesized the single-ion monomer (SIM) with an attached anion incorporated into the structure to facilitate lithium cation motion. I anticipate copolymerizing this SIM with ONDI-12 at lower SIM percentages using ring-opening metathesis polymerization (ROMP) and subsequently measuring their conductivity. Identifying the SIM to ONDI-12 ratio that optimizes conductivity will improve our understanding of these materials and potentially advance future polymer electrolyte design.

One of the most prevalent issues in regenerative medicine is the impact of spinal cord injuries, as it can lead to an irreparable buildup of inhibitory scar tissue and, thus, paralysis. However, organisms such as Xenopus tropicalis tadpoles are able to regenerate their tails as soon as one week post-injury. By studying how they successfully regenerate, we can start to generate effective therapies for spinal cord medicine. I specifically want to know how quickly neurons populate the regenerating spinal cord and how this repopulation leads to functional motor recovery. To do this, I used the process of immunohistochemistry, where a fluorescent marker antibody binds to specific cells to create a fluorescent image for visualization purposes. First, I amputated around ⅓ of their tail and created clutches of tadpoles stained for a neuron-specific protein. From my imaging, I noticed that the neurons populated the regenerating spinal cord by five days post-amputation (dpa). I became curious about how this regeneration rate impacted their ability to swim. To test this question, we set up a camera with a lightbox to set up Petri dishes of tadpoles. Then, I uploaded recordings of their swimming into a platform called ImageJ to use particle tracking to quantify the paths of each tadpole into measures such as distance, displacement, and velocity. Currently, we are trying to find other antibody markers that can provide more specific staining of neurons so the program can count them. With more specific staining, I hope to count the number of neurons over a set of zero, three, five, and seven dpa tadpoles. This project will help us answer foundational questions about how Xenopus tropicalis tadpoles regenerate functional neurons after injury.

My research is an analysis of the pervasive issue of gender-based violence faced by women in India. Despite the presence of constitutional safeguards, the entrenched patriachal norms, and the prevalent misogyny in Indian society continue to deny women the healthy and peaceful lives they rightfully deserve. By analyzing pre-existing data along with data collected from interviews conducted with women between the ages of 35-45 from village Gumtala in Punjab, I have conducted a study to examine the relationship between postponed age of marriage and gender-based violence. It's through the lens of these women, I argue that decades of suppression in the name of culture have normalized violence against them. I conducted this study with careful consideration, ensuring that the questions posed in the semi-structured interviews were sensitive to the unique backgrounds and experiences of the women involved. Through the method of convenience sampling and qualitative analysis of interview data, I identified patterns of gender based violence and socio-economic factors. The results of my study suggest that early marriage not only increases women's vulnerability but also fosters economic dependence and social isolation of young brides that fuels the act of dowry practices causing marital abuse.These findings are not only essential in creating awareness regarding the vulnerable state of these women but also help me pose possible policy interventions by the Governmnet of India. My research has allowed me to argue how raising the legal marriage age for women to 25 mitigates gender-based violence, allowing women more time to pursue education and build financial independence before marriage. This study is an ode to all the women who are denied a voice.

Sulfate aerosols cause pollution and affect climate by influencing cloud properties and incoming solar radiation. Emissions and abundances of sulfur-containing aerosols are one of the largest sources of uncertainties in global climate modeling. The largest biogenic and most uncertain emission source of sulfur aerosols is from phytoplankton in the form of dimethyl sulfide (DMS). In the atmosphere, DMS is oxidized to methanesulfonic acid (MSA), sulfur dioxide, and hydroperoxymethyl thioformate (HPMTF), all of which can form sulfate. Historical emissions of DMS are studied by measuring MSA concentrations in ice cores as a proxy for DMS oxidation. Declining levels of MSA have been found in ice core records, implying that production of DMS has also been decreasing; however, anthropogenically driven changes in atmospheric chemistry have altered the ratio of MSA to sulfate produced from DMS over time. To better understand DMS oxidation mechanisms and its relationship to the production of MSA and sulfate aerosols, we need more recent ice core records of MSA and sulfur isotopes of sulfate (δ³⁴S(SO₄^2–)) at higher temporal resolution. To measure δ³⁴S(SO₄^2–) at monthly resolution in an ice core, the measurement size is smaller than previously measured by an order of magnitude, at about 1 µg S per sample. We will develop a method to isolate 1 µg of sulfur from an ice core sample by concentrating the sulfur using an anion-retaining resin, precipitating with barium chloride, and drying in an oven. We will quantify the efficacy of our method using a stable isotope mass spectrometer compared to laboratory-prepared standards. We expect that we will reduce our sample size by an order of magnitude (to 0.1 μg sulfur) and improve the accuracy by 50%. Quantifying sulfur isotopes at this resolution will provide information about the seasonality and change in phytoplankton sulfate production.

Every forty seconds in the United States, someone suffers from a heart attack or stroke. Heart attack and stroke can be caused by the blockage of blood vessels by small, transient emboli, or clots. The risk of these attacks increases with age, and there are sex and ethnic based inequalities in the prevalence of these thrombotic events. These emboli can form through various pathways. One factor that can affect platelet aggregation is Von Willebrand Factor, which acts as a scaffold for platelets to bind to, creating a stable clot. In our microfluidic devices, I have observed the detachment of small emboli from large platelet aggregations, which I believe can model transient thrombotic embolisms in the body. Von Willebrand Factor is of specific interest to me, as I have observed that higher levels result in larger platelet aggregations, and therefore more emboli detachment events. This project investigated the question: What is the composition of these detached emboli and what part does Von Willebrand Factor play in their makeup? I hypothesized that these emboli will have high Von Willebrand Factor content and a core of activated platelets surrounded by inactivated platelets, which is what allows for the emboli’s detachment and transient nature. I employed flow cytometry, which causes specific components stained by fluorescent antibodies like PAC-1 and P-selectin to light up. This allows me to determine the composition of the emboli in terms of activated platelets and Von Willebrand factor. If my hypothesis that the emboli have high Von Willebrand Factor content is correct, this could have significant implications for treatments for heart attack and stroke, as current antiplatelet therapies do not target Von Willebrand Factor. If we can create more efficient treatments by knowing what the treatments should be targeting, there are countless people whose health can be positively impacted.

Each winter, the northeastern U.S. experiences powerful storms that cover cities in snow and ice, which result in millions of dollars in damage, halt travel, and disrupt essential services. Yet, the type, intensity, and distribution of precipitation is unique to each winter storm. This research project aims to provide a greater understanding of the precipitation properties and distribution in snowstorms, through focusing on a major winter storm that occurred over the Midwest on 17 February 2022 and was the target of a research flight conducted during the Investigation of Microphysics and Precipitation for Atlantic Coast-Threatening Snowstorms (IMPACTS) field campaign. Radar data collected during this research flight provides a unique perspective of the vertical cloud and precipitation structure, and numerical model fields provide the environmental context of the structures observed in the radar measurements. This storm had a frontal boundary, or a strong thermal contrast, that provided lift needed for the production of precipitation and had sub-freezing temperatures so that the precipitation fell as snow.This frontal boundary consisted of warm air originating from southern latitudes riding over colder air originating from northern latitudes. Analysis of the vertical cloud and precipitation structure from radar data and the in situ cloud particle measurements collected during the flight revealed that regions of higher reflectivity had larger particles and greater ice water content, compared to regions with lower reflectivity. The analysis also includes examining how the cloud particle properties are different depending on the origin of the air masses (from the north or south) that form the storm. By relating the temporal and spatial information regarding the air masses to the high-resolution radar and microphysics data collected by the IMPACTS airborne instruments, the results of this analysis will ultimately support increasing the accuracy of snow prediction.

Mirrors have been a contextually important part of architecture and painting since before the Renaissance, but during the Baroque and leading into the Rococo, mirrors became a central aspect to power dynamics in built environments and were utilized as a tool to control self-representations. This paper investigates the ways in which mirrors were activated in interior architecture and paintings, as well as how they underscored the “soft” power of elite women during the Rococo period by analyzing François Boucher’s Madame de Pompadour (1756) as an example of mirrors and expressions of the self and purposeful manifestations of “soft” power. I place Boucher’s painting in the context of contemporary interior spaces, such as the Versailles’ Hall of Mirrors (1678) which illustrates “hard” political power and the Salon de la Princesse room in Paris’ Hôtel de Soubise (1732) which communicates a softer power, as examples to how mirrors enforce performative power in spaces. I explain how power dynamics are reinforced through interior spaces paying special attention to use of space, decoration/motifs, and the location/orientation of the interior architecture and design and exploring Madame de Pompadour’s use of mirrors to subtly control a narrative that elevated her status in the French court.
The implications of this work recontextualize the presence of mirrors in paintings of the Rococo period and give a new view on the roots of the subtly psychological implications of mirrors within the built environment. Mirrors as a topic of specific study within aristocratic interiors lacks robust research, my work aims to fill part of this gap by seeking to understand how mirrors established themselves within architecture through power preformances and psychology.

Ford Madox Brown's 1865 painting Work depicts a group of laborers digging a sewage line on a busy London street. Widely considered the most important painting of Ford Madox Brown’s career, Work is also one of the most radical paintings of the pre-Raphaelite period. In this presentation, I will argue that the dignity afforded to manual laborers in Work establishes a pride in British identity and a push for social reform in line with Christian socialist values. I will support this argument by analyzing the painting's composition, use of symbolism, and historical context. In the painting’s composition and content, the work of William Hogarth and the Pre-Raphaelite Brotherhood reveal themselves as influences on Brown’s art and politics. I will also discuss how other scholars have interpreted Work’s symbolism and sociopolitical context, especially the writings of John A. Walker and Jenny Plastow. Through the painting and Brown’s accompanying catalog, Work instructs the importance of education and Christian values to social reform. Work is a complex and historically significant painting that celebrates labor during a turbulent time in England’s history. This paper will strengthen understanding of Work’s political rhetoric and symbolism, and in doing so illuminate the context of its creation.

To perform its function as a tumor suppressor, breast cancer 1 (BRCA1) must dimerize with BRCA1-associated RING domain protein 1 (BARD1). Due to this critical interaction, pathogenic BARD1 variants are also associated with increased breast and ovarian cancer risk. Genetic testing has identified many rare single-nucleotide variants (SNVs) that cause missense amino acid substitutions in BARD1. Currently, 93% (1,692 of 1,819) of BARD1 missense SNVs are classified as a variant of uncertain significance (VUS) in ClinVar. A VUS classification prevents clinicians from using genetic test results to guide patient care. Consequently, there is a strong need to functionally assess BARD1 SNVs to help resolve VUS. We applied a multiplex assay for variant effect called saturation genome editing (SGE) to functionally assess all possible 12,000 SNVs and 2,300 3-base deletions in BARD1. In SGE, we use CRISPR-Cas9 to edit all possible SNVs into a region of BARD1 in haploid HAP1 cells. BARD1 is essential for cell growth, therefore cells edited with loss-of-function variants become depleted from the population. We track which SNVs become depleted from the population by sequencing. We then generate functional scores for each variant by calculating the change in the abundance of a variant in the original SNV library versus its abundance in the cell population after 13 days in culture. Thus far, I have generated reagents for all 14,300 variants and 2,400 have completed the full experimental pipeline. Functional scores for the functionally critical BRCA1 interaction domain show depletion of 94% stop-gain, 48% splice-site, and 21% missense variants relative to 5% synonymous and 6% intronic variants. This ultimately demonstrates SGE’s ability to accurately identify functionally normal and loss-of-function BARD1 variants. Generating functional scores for all possible BARD1 variants will provide the functional evidence needed for reclassifying BARD1 VUS and definitive test results for providers treating patients with BARD1 variants.

The human genome is associated with numerous variations, some of which can be pathogenic. Any variations larger than 50 base pairs (bp) are considered structural variants (SVs), and SVs impacting copy number of the genome are termed copy number variants (CNVs). By studying CNVs, we can understand the underlying mechanisms of damage and repair within DNA, which can help work towards prevention and cure in other fields, such as cancer genomics. My project investigates a CNV on chromosome 2 of a pediatric patient, who presents with tetralogy of Fallot, global development delay, and multiple other congenital anomalies. Using array comparative genomics hybridization (aCGH), we identified a 3.2 megabase (Mb) complex genomic rearrangement (CGR) spanning the 2q31 region of the proband’s chromosome 2. Detailed analysis of the short-read whole genome sequencing (WGS) allowed us to locate the exact coordinates of each junction, or the beginning and end points of each extra copy. PacBio long-read genome sequencing and optical genome mapping detected the same junctions and facilitated confirmation of the overall structure. The CGR can be characterized as a duplication-triplication-duplication-triplication-duplication (DUP-TRP-DUP-TRP-DUP), meaning this segment of the genome contains a segment of alternating 1 and 2 extra copies of this region. The CGR is de novo, or not inherited from the proband’s parents. Due to the nature of this variant, it is likely to be impacting the phenotype of our patient. To establish a genotype-phenotype correlation, I did a literature review of patients with overlapping CGRs, comparing their phenotypes to our proband, as well as reviewing any known disease-associated genes in the region using the online catalog of human genes and genetic disorders (OMIM). Patient and disease comparison revealed the extreme rarity of our patient’s CGR, leading us to believe the phenotype of the proband results from impact of multiple genes in the affected region.

In order to design effective countermeasures against HIV, we must first understand the forces that drive it to evolve resistance within hosts. While linkage patterns in genetic data are potentially a powerful tool to quantify the relative contributions of multiple evolutionary forces (mutation, recombination, selection) acting during an HIV infection, the severe viral population bottlenecks accompanying drug therapy complicate these patterns. To interpret genetic linkage in the context of such major changes in population structure (which are themselves driven by specific mutations), we develop a simulation framework for viral evolution in which genetics and population structure influence each other. This framework overcomes limitations from both dynamical modeling, in which patterns of linked variation are ignored, and from population genetic modeling, in which population structure is predetermined. Using few parameters, we are able to reproduce linkage patterns and population bottlenecks that broadly conform to those observed in vivo. As a case study to demonstrate this model’s utility, we consider a recent hypothesis that viral recombination is suppressed during population bottlenecks due to diminished opportunities for coinfection. In simulating populations with and without recombination suppression during population contraction, we show that this effect measurably changes genetic diversity in rebounding populations, but is less visible when examining simulated viral loads or resistance mutations alone. Then, using this model as a null expectation for linkage patterns, we assess if the linkage structure in HIV populations treated with bNAbs is consistent with density-dependent recombination in vivo. Collectively, our work demonstrates that, by generating realistic null expectations of linkage under complex changes in population structure, we can employ linkage patterns as a powerful source of information for evaluating viral evolutionary hypotheses.

This paper seeks to find and explain the intersection between the Irish War for Independence (1919-1921) and Irish Rebel Music recorded between 1960 and 2017. Ireland gained independence from the British Empire in 1921, but the bitterness between the two nations remained. That tension became more prevalent in the 1960s and continued for the decades to come. Those decades became a time for Irish musicians to flourish. For the Irish, using music in a political sense is nothing new. Music has always played a large part in Irish society. Throughout the eight hundred years of oppression from the British Empire, the Irish always continued to play on. This paper examines the lyrics of Irish rebel music recorded between 1960 and 2017 that reflect the events of the Irish War for Independence and the lasting effects the war had on Irish society. The question driving this research is how Irish rebel music, between 1960 and 2017 memorializes the Irish War for Independence. By comparing the literature surrounding the War for Independence and Irish Music, this paper argues that the intersection between the events of the Irish War for Independence (1919-1921) and Irish rebel music (recorded between 1960 and 2017) reveals the major influence of music within Irish society in the late twentieth century. Irish musicians used music and lyrics not only to fight the British Empire but to also build a proud and strong nation.

In response to the nuclear discharge incident on 21st August 2023 from the Fukushima nuclear reactors into the Pacific Ocean, Japan found itself at the forefront of a renewed global discourse on nuclear energy policy again post the 2011 Fukushima Daiichi accident. The consequential surge in antinuclear sentiment, particularly noteworthy within the East Asia region, serves as the impetus for this research, which seeks to meticulously analyze the potential impact of such sentiment on the public's perception of Japan's nuclear energy policy. This research adopts Akhiro Ogawa's conceptual framework of "anti-nuclear citizen" to scrutinize its influence on the youth demographic, and whether or not the identity influences their attitude towards Japan's current pro-nuclear policy. To ascertain empirical data, the survey method is employed to measure college youth’s sense of precarity, political involvement, and conscientiousness, serving as indicators of one’s alignment with the antinuclear citizen identity. The survey targets undergraduate Japanese exchange students and domestic students who are interested in Japanese studies at the University of Washington and undergraduate students in Japan and aims to delineate the nuanced correlations between antinuclear citizenship and specific policy perceptions. Recognizing the survey’s limitation of snowball sampling predicts a collective behavior that may not be representative of the general population, the study acknowledges potential biases and would like to serve as evidence to elucidate the presence or absence of “antinuclear citizens” within the defined focus group. Furthermore, the survey results are anticipated to delve into the rationales behind certain policy perceptions: generally anti-nuclear or pro-nuclear, leveraging participants' international experiences to augment the evidential support for the application of Ogawa's "antinuclear citizen" ideology. By analyzing the attitudes of the surveyed population toward Japan's nuclear policies, this research aspires to contribute to the conversation of the complex interplay between antinuclear sentiment, identity, and policy perceptions.

This digital humanities capstone project builds upon my senior thesis “An Analysis of Western Perspectives on the Khmer Republic, 1970-2023”. The thesis examined Western scholarly works related to the escalating events that enabled the Khmer Rouge’s rise in Cambodia, revealing biases in how the events leading up to the Cambodian Genocide were portrayed. This research demonstrated that the roots of the Cambodian genocide stretched back years before the Khmer Rouge came to power, fueled by political instability and civil war. Through an interactive digital timeline, this project synthesizes primary and secondary sources across media reports, government records, and academic analysis, to name a few, to visually display the narratives and divergences in Western scholar’s perspectives. My main research question has changed from the start of this project, today resulting in: How can a digital timeline effectively showcase the divergences in Western scholars’ portrayal of events during the Lon Nol Era, that preceded the Cambodian Genocide? Over the past two semesters, I have been building a website to illustrate the history leading up to the Khmer Rouge regime. The website features an interactive timeline and globe, based on latitude and longitude points. It features three interlinking sections tracking: 1) Scholarly Works, 2) Surrounding World Events, and 3) The Lon Nol Era and Cambodian Genocide. By revealing biases and gaps through a visual model, it can reveal blind spots or skewed narratives. It can also track interconnections, and observe how scholarly interpretations evolved to provide context for the escalating political instability to demonstrate the Khmer Rouge’s rise in power. By uniquely challenging oversimplified narratives, this project can provide a more contextual understanding of how Western perspectives shaped understanding of the Cambodian Genocide.

This paper quantifies the impact on the South Korean Cultural Entertainment Industry (CEI) after China's "Korea Limitation Order". In November 2016, Beijing restricted South Korean artists to hold concerts and the broadcast of South Korean TV dramas, also banned cooperation between the two countries in the entertainments industries. This paper examines the effect did this had on exports of cultural products from Korea, and how Korea was able to adjust to this Geopolitical shock. I examine exports from Korea, to China, Japan, and the United States: Data from Cultural Entertainment Industry Products Exports, tangible or intangible goods that can create economic added value, such as Korean music and Filming Industries, and South Korea inbound foreign tourism statistics during 2011-2019 were collected. Deriving the deviation from an estimated autoregressive moving average (ARMA) model specification of exports from Korea after the “Korea Limitation Order,” in Difference-in-Difference (DID) Model, I found that South Korea's tourism industry has been particularly affected, with the most significant decline compared to the other two countries. At the same time, in the tourism industry, the substitution effect of Japanese market for the Chinese market is clear to see. The film and broadcasting industries have seen similar but less dramatic declines than tourism industry. There is no evidence that sectors of the cultural industry that are not included in this restriction category such as cartoon industry, are impacted by this shock.

The mechanisms guiding the sensory detection of pain and the subsequent sensitization of damaged tissue to mechanical and thermal stimuli are relatively well understood. However, mechanisms guiding the transformation of nociception into the negative feelings associated with pain remains largely unknown. This affective component, notably in chronic pain, translates into an intense emotional impact on patients and can contribute to the development of comorbid psychiatric disorders. The elderly population has a propensity to be socially isolated and face exacerbated effects of chronic pain. In 2021, an estimated 20.9% of U.S adults suffer from chronic pain with persons over 65 years of age having the greatest propensity of acquiring the disease. Due to this, clinical intervention models call for a more holistic approach to pain intervention that incorporates lifestyle and nutritional factors, extending beyond pharmacological treatments. One of these promising non-pharmacological interventions is positive social interaction, which has been shown to alleviate pain and suffering. Several studies show that humans who maintain strong social bonds recover from injuries faster than people without them. However, it has not yet been evaluated the extent to which this phenomenon occurs in geriatric animals and its relative efficacy as a social intervention to alleviate chronic pain in injured mice. My project seeks to gauge whether social intervention can alleviate chronic pain symptoms in aged mice and to unveil the underlying mechanisms guiding these successful non-pharmacological treatments. I will achieve this through two aims: an evaluation of social self administration as an intervention for chronic pain, and histological analysis to identify gene expression changes as a result of social interaction. Future research will include mini-scope endomicroscopy recordings to visualize communication among major brain regions, and comparison of cell ensemble activity between groups of mice will lead to the identification of relevant neural ensembles and molecules.

In mammals, circadian rhythms are regulated by a hierarchy of oscillators governed by a central circadian pacemaker in the suprachiasmatic nucleus (SCN), which is principally entrained by the light-dark (LD) cycle. Recent experiments in our lab have revealed that cyclic 24-h fearful stimuli can act as a potent nonphotic zeitgeber, entraining circadian rhythms of behavior in mice and rats. This discovery utilized a naturalistic rodent cage with a safe nesting area separated from a foraging area where feeding and drinking occur. While foraging behaviors naturally occur at night, when the foraging area is rendered dangerous by nocturnal aversive stimuli (footshocks), animals entrain behaviors to the shock schedule by shifting activity to the daytime. Under conditions of fear-entrainment, SCN clock gene expression remains loyal to the LD cycle and the SCN is necessary but not sufficient for sustaining diurnal activity. Therefore, we propose the existence of extra-SCN fear-entrained oscillators capable of overriding SCN output and influencing behavioral timing. Here, we subjected 16 mice to either diurnal shocks (DS; control) or nocturnal shocks (NS) under a 12:12 LD cycle. Following confirmation of fear-entrainment, animals were released into constant conditions and sacrificed between 24-36h after the last presentation of footshocks, either CT 1 or CT13. Brains were dissected, sliced, prepared for immunohistochemistry processing, and c-Fos protein quantification is currently underway in the SCN, basolateral amygdala, paraventricular nucleus of the thalamus, and dentate gyrus. We hypothesize that c-Fos expression within the SCN will align with the LD cycle, while centers involved in fear processing and memory will exhibit altered levels of c-Fos expression in response to time-specific fear. Results from this study may be useful for identifying putative brain regions containing fear-entrainable oscillator(s).

Chimeric Antigen Receptor T (CAR T) Cells are receptor proteins that can be modified to allow T cells to target specific antigens. CAR T therapy has shown promise in preclinical experiments in patients with solid tumors, such as glioblastoma, however limitations in distribution of CAR T cells in the brain limit the effectiveness of this treatment. Most commonly, CAR T cells are administered intraventricularly through a surgically implanted device and allowed to diffuse throughout the cerebrospinal fluid filled cavities and pathways to reach the tumor. However, there is little evidence supporting the effectiveness of current methods of administration, potentially due to a lack of target engagement. We hypothesize that the glymphatic system of the brain could be used to optimize delivery of CAR T cells to solid tumors. The glymphatic system is a network of perivascular pathways that facilitates the anatomically distinct movement of cerebrospinal fluid (CSF) into the interstitium of the brain, helps distribute solutes such as glucose, lipids, and neurotransmitters, and serves as a solute clearance system in the brain. Physiologically, glymphatic function is mediated by different factors such as arterial pulsation, vasomotion, and heart rate – which can be manipulated with anesthetics, pharmaceuticals, or even sleep. We proposed exploration of the difference in parenchymal distribution of CAR T cells when injected in the cisterna magna versus intraventricularly in mice. Non-tumor bearing mice were injected via the cisterna magna or intraventricularly with fluorescently labelled CAR T cells. We observed that at 1-, 4-, and 24-hours post-injection, CAR T cells were localized in the sub-ventricular regions similarly, regardless of injection site. In follow-up experiments, we will employ the same technique in tumor bearing mice, with and without pharmacological intervention to define the effect of glymphatic function on distribution and effectiveness of CAR T cells.

Androgen Deprivation Therapy (ADT) is the standard treatment for advanced prostate cancer (PCa), although it adversely affects muscle mass, physical function, and quality of life (QOL). It is unknown whether 1) these factors Pre-ADT can predict ADT-induced changes or 2) if non-White, non-Hispanic (NWNH) men experience greater adverse impact on these outcomes than White, non-Hispanic (WNH) men. I hypothesized that 1) greater muscle mass Pre-ADT would be protective against ADT-induced changes in muscle mass, physical function, and QOL and 2) NWNH men would experience worse changes in muscle mass, physical function, and QOL than WNH men. I assessed lean body mass (LBM) [dual energy x-ray absorptiometry], physical function [aerobic capacity (VO2Peak), hand grip strength (HGS), 6-minute walk test (6MWT), stair climb power (SCP)], and QOL [QLQ-C30 questionnaire] in PCa patients (n=59) from the Seattle VA before and 6 months after ADT. I used Pearson correlations to test associations between variables and independent t-tests to compare variables between WNH and NWNH men. Larger LBM (r=0.33, p=0.019, n=50), lower QLQ-C30 Function (r=0.31, p=0.03, n=50), and higher QLQ-C30 Fatigue (r=0.40, p=0.004, n=50) Pre-ADT were correlated with larger 6-month decreases in LBM. NWNH men (n=20) displayed significantly worse Pre-ADT HGS (p=0.038), 6MWT (p=0.037), and SCP (p=0.005) than WNH men (n=39). I anticipate that NWNH men will display worse 6-month changes in LBM, HGS, 6MWT, SCP, and QOL than WNH men in my ongoing analyses. Contrary to my hypothesis, larger LBM Pre-ADT was not protective against muscle loss. Consistent with reports that NWNH men experience greater tumor-related adverse impacts of PCa treatment, worse functional performance Pre-ADT may indicate physical function of these individuals is also disproportionately adversely impacted by ADT. By identifying predictors of adverse ADT outcomes, researchers can develop interventions aimed at preserving muscle mass, physical function, and QOL during ADT for high-risk populations. Additionally, reducing the disproportionate adverse effects of ADT on NWNH men will address disparities within PCa treatment, promoting personalized healthcare optimizing outcomes for all patients. 

6PPD-quinone (6PPDQ), a transformation product of an anti-oxidant used in tire manufacturing, was recently identified as the causal agent of acute mortality in coho salmon. Abrasion on tires by road surfaces create tire wear particles (TWPs). Both TWPs and the accumulation of waste tires pose risks of leaching 6PPDQ into stormwater runoff. Crumb rubbers, which are manufactured to reduce landfill tire waste and applied in turf infills, may also leach 6PPDQ. My research aims to determine the conditions at which crumb rubber can be pyrolyzed to prevent 6PPDQ leaching from tire recycling options. If pyrolysis successfully removes 6PPDQ from crumb rubber, then the resulting material can be applied as an absorbent tire char to remove contaminants from water. Waste tire crumb rubber samples were pyrolyzed in a tube furnace under nitrogen flow for 90 minutes at a range of different temperatures. Methanol-based solvent extraction was used to extract the remaining 6PPDQ from the pyrolyzed samples and diluted until suitable for liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis. It is observed that as the pyrolysis temperature increases, the mass of 6PPDQ leached from pyrolyzed crumb rubber decreases. The results of this study allow us to understand the limitations of pyrolyzing tire rubber to develop activated carbon. To further investigate the feasibility of waste tire activated carbon, a chemical activation step will be added in pyrolysis to better replicate the creation of activated carbon.

The tumor microenvironment (TME) is a complex network of cells surrounding the tumor including blood vessels and immune cells, such as dendritic cells (DCs) and natural killer (NK) cells. DCs play a key role in driving anti-tumor immune responses against cancer. We recently discovered that NK cells control immunotherapy responses through their production of a cytokine called FMS-like tyrosine kinase 3 ligand (Flt3L), which supports DC function. However, the mechanisms regulating NK cell production of Flt3L in the tumor remain unknown. Preliminary work from our lab has suggested that hypoxia, a common feature of solid tumors marked by reduced oxygen levels, diminishes Flt3L production by NK cells. We hypothesize that spatial localization of NK cells within the hypoxic and normoxic TME differentially shapes the NK cell – DC axis. Two mouse melanoma tumors, highly hypoxic YUMM1.G1 and minimally hypoxic YUMM1.7, were grown in XCR1-venus mice (genetically modified to visualize DCs). 1.5 hr prior to tumor taking, I injected mice with the hypoxia marker pimonidazole, a compound which covalently binds to thiol groups in proteins and amino acids under hypoxic conditions. I am using a multiplex immunofluorescence panel to study NK cell (anti-NKp46), DC (anti-Venus), blood vessels (anti-CD31) and hypoxia (anti-pimonidazole) localization in the TME. By mapping the TME, I aim to uncover the spatial distribution of hypoxia and its impact on the abundance and distribution of NK cells and DCs. I hypothesize less colocalization of NKs and DCs in hypoxic regions compared to normoxic regions due to reduced NK cell function. My research seeks to explore the interplay between hypoxia and immune cell localization, potentially showing hypoxia to be a tumor-intrinsic regulator that shapes the NK cell-DC axis. Overall, this will improve our understanding of immune cell behavior within the TME, and predict new therapeutic treatments to improve patient outcome.

Preterm birth is one of the leading causes of infant morbidity and mortality worldwide, with a strong association between the degree of prematurity and the likelihood of death or neurodevelopmental impairment. Intracranial hemorrhage (ICH) is one of the most common neurological injuries for extremely preterm infants (born less than 28 weeks’ gestation). During the last trimester of pregnancy, neurons and glial cells develop in the germinal matrix requiring vast amounts of vascular support. In preterm infants, disturbances to blood and hydrostatic pressure are thought to rupture the immature vessels of the germinal matrix, leading to the bleeding in and around the ventricles. ICH is rated on a scale of I to IV, with severe ICH being grade III-IV. Mortality associated with ICH ranges from 30-60 percent, increasing with ICH severity, and survivors have an increased risk of cerebral palsy, seizures, and neurodevelopmental delay. From 2018-2020 the University of Washington (UW) neonatal intensive care unit (NICU) implemented an ICH Prevention Bundle, which focused on minimizing blood pressure disturbances during the first 72 hours after birth in infants born extremely premature, and appeared to result in a decrease in severe ICH. This study will evaluate the incidence rate of ICH at the UW NICU over a ten-year period. In a retrospective analysis of the UW NICU’s admissions, we will investigate extremely preterm infants born during the time periods of December 2013-September 2016 versus January 2017-December 2023 and record the incidence of ICH. Our primary outcomes will be ICH, by grades I-IV, as well as ICH complications such as posthemorrhagic ventricular dilatation with and without need for intervention, and death before discharge. We hypothesize that with improved prevention methods, such as the implementation of the ICH Prevention Bundle, we will see an associated long-term decrease in the incidence rate of ICH.

Gastrointestinal symptoms are a common comorbidity of autism spectrum disorder (ASD), and individuals with the disorder tend to have a distinct gut microbial community composition and circulating metabolomes. My work in the Rajakovich Group focuses on a gut-derived metabolite, 4-ethylphenolsulfate (4-EPS), found in higher abundance in ASD mouse models and children with ASD. 4-Ethylphenol (4-EP), its precursor, is produced by gut microbiota before host-mediated sulfation, but the microbial biosynthetic pathway is unknown. A proposed metabolic pathway suggests the microbial stepwise conversion of plant-derived complex polysaccharides to 4-EP. My project goal is to identify a gut microbial enzyme responsible for the first step of this proposed pathway: a hydroxycinnamoyl esterase. I used literature searches and bioinformatics tools to identify characterized bacterial cinnamoyl esterases and candidate enzymes. I designed plasmids for two candidate enzymes (both from E. faecium, known to colonize the gut) and one characterized esterase (from L. plantarum). Currently, I am working on expressing the proteins in E. coli cells and purifying them by affinity chromatography. Once purified, I will assess the enzymes for their anticipated cinnamoyl esterase activity by incubating them with dietary hydroxycinnamic acid esters and detecting products with high-performance liquid chromatography (HPLC) and UV/Vis spectroscopy. Since the candidate enzymes are homologs of confirmed esterases and have conserved catalytic motifs, I hypothesize that they will have hydrolytic activity. If correct, I will see consumption of the substrate (no detection) and detect the anticipated products. Positive results from these assays would complement ongoing work by the lab to identify other E. faecium enzymes in this proposed pathway. Though it is debated if 4-EPS is causal to the disorder or simply a biomarker, elucidating its biosynthetic pathway and studying the biochemistry of gut microbes will contribute to detangling the gut’s role in ASD.

Graph workloads are challenging for query optimizers in databases because of query features like larger sizes, frequent joins, and fewer filters. Traditional methods see large errors on queries with more joins, while machine learning methods tend to be complex and slower. We propose a framework to improve estimators by using graph colorings to make compact summaries of a data graph, storing important information about node relations. By modelling cardinality estimation as a subgraph matching problem, we can make use of this summary information and traverse the lifted graph to estimate the number of query graph matches. Additionally, we explore optimizations such as node summation and sampling to enable estimation even for larger queries. After evaluating various designs using this framework, we find improvements up to 100x to cardinality estimation accuracies compared to other recent methods while still maintaining efficient runtimes and memory usage. We discovered that quasi-stable colors, where nodes of one color have similar connections to other colors, result in these improved results when used to build the summary. These findings help improve graph database performance and offer a new application for graph theory.

Computational protein design has successfully designed nanoparticle cages that self-assemble and effectively deliver encapsulated therapeutics to cells. These nanoparticle cages are readily taken up by the cell via receptor-mediated endocytosis. Despite the promise of these cages, one of the greatest challenges that remain is the successful endosomal escape of the encapsulated biologics and their precise delivery to the cytosol. To address this, we have engineered a high throughput complementation assay, based on split green fluorescent protein (GFP) construct, that helps screen and quantify cytoplasmic delivery of therapeutics through fluorescence intensity. Split-GFP is a protein complementation assay in which the normally monomeric GFP is made of two fragments: the larger non-fluorescent beta barrel and a 15 amino acid (a.a) peptide. When these two components unite, the GFP fluoresces. In this project, I created a stable HeLa cell line expressing the beta barrel of split GFP using lentiviral transduction under antibiotic selection. The cell line has been further validated, through transient transfection of the complementary 15 a.a peptide to test the assay performance. I propose to test endosomal escape, through introduction of endolytic peptides (EEPs) into model proteins, which force early endosomal membrane fusion and destabilization. Future research will explore adapted designs of nanoparticle cages, incorporating the EEPs and the split-GFP complementary strand in the HeLa cell line, to quantify the endosomal escape of our designs. The outlook of this project has transformative implications for targeted therapeutic delivery. By creating a screening assay that can quantify targeted delivery into cytosol, we can expedite refinement of protein designs for therapeutic delivery, thus accelerating the timeline for developing novel protein-based therapeutics.

The University of Washington Neonatal Intensive Care Unit (NICU) Extremely Preterm (EP) program aims to provide outstanding care to EP infants born at 22 0/7-24 6/7 weeks’ gestation. The objective of this study is to assess the feasibility of night telerounds in a level IV academic NICU for EP neonates to enhance the support for teams caring for EP patients. This was a retrospective cohort study. Through review and analysis of electronic medical records, the EP database, and telemedicine records from July 2022 to June 2023, the frequency and duration of telerounds during the night shift (8-11pm) were examined. Comparisons of EP outcomes were made between two epochs: 6 months pre- and 6 months post-telerounds implementation. Descriptive statistics, chi square and independent samples t-test were used to compare EP outcomes between epochs. There were a total of 195 telerounds encounters, with improvements noted in network connectivity over time (fewer dropped calls lasting <20 sec). In the pre-implementation period, 9 (36%) of EPs were transferred for subspecialty/surgical care, mortality before 36 weeks’ postmenstrual age (PMA) was 14 (56%) and median length of stay (LOS) for survivors to discharge was 182 days (SD 72). Post-implementation, 10 (50%) EPs were transferred with mortality before 36 weeks’ PMA of 7 (35%) and median LOS for survivors to discharge was 139 days (SD 24). In conclusion, there was a reduction in neonatal mortality before 36 weeks’ postmenstrual age and a trend towards decreased length of stay during the post-implementation period. The findings suggest that night telerounds are feasible in supporting bedside NICU teams caring for critically ill EP neonates and may facilitate care advancement. Additional chart review is in progress to characterize orders entered during night rounds in order to evaluate the impact of this intervention.

Chronic kidney disease of unknown etiology (CKDu) is a pervasive condition not prompted by diabetes or hypertension but instead by environmental stimuli and occupational associated risks. Often detected only in advanced stages, CKDu necessitates interventions such as dialysis and kidney transplant, significantly burdening healthcare systems globally and disproportionally affecting rural populations. Ochratoxin-A (OTA), an abundant, ubiquitous, natural contaminant found in food products and is among the postulated risk factors for (CKDu). While animal studies indicate dysregulation of mitochondrial dynamics and production of superoxides via redox cycling as potential mechanisms of OTA-associated nephrotoxicity, its exposure risk in humans and kidney health remain poorly understood. This study investigates the biological pathways that contribute to the toxicity of OTA in the proximal tubule, leading to CKDu. Luminescence-based imaging approach evaluated the occurrence of OTA-induced oxidative stress in human proximal tubular epithelial cells (PTEC) by detecting cytoplasmic reactive oxygen species (ROS). Preliminary mRNA transcriptomic analysis has indicated the down-regulation of glutathione pathways, a major antioxidant pathway removing cellular oxygen. I probed OTA-treated PTECs with a reduced glutathione detection reagent, ThiolTracker Violet, to investigate the cell’s response capability for oxidative stress and detoxify xenobiotics. To verify oxidative stress mediated by OTA in live cells, CellROX Green Reagent probe treated PTECs were brought under confocal microscopy to visualize mitochondrial phenotype. The results of this investigation seek to reveal the metabolic response to OTA cytotoxicity in the human kidney and elucidate its role in CKDu progression to address diagnostic challenges and confront unmet medical needs.

Spinal cord injury (SCI) causes significant sensorimotor deficits that negatively impact autonomy and quality-of-life. In a previous study, we determined that optogenetic spinal stimulation significantly enhanced forelimb recovery, axonal growth, and angiogenesis compared to sham controls. However, we have yet to determine the synaptic changes associated with optogenetic stimulation after SCI. The current project addresses this important detail by quantifying the synaptic changes that occur with and without optogenetic spinal stimulation in rats with cervical SCI.To investigate this, rats received a moderate hemicontusion of the 4th cervical segment (C4) and a spinal injection of an optogenetic viral vector (AAV2-hSyn-ChR2-YFP) to express light-sensitive proteins in the ipsilateral sixth segment (C6). Four weeks later, rats received a second surgery to receive a blue uLED implanted over ipsilateral C6 for optogenetic or sham stimulation. Rats were trained and scored regularly on a variety of forelimb behavioral tasks throughout the course of the study, while the rats in the stimulated group received stimulation 1x/week for 6 weeks beginning on the 6th week post-injury. After perfusion, the cervical spinal cord was sectioned and underwent immunohistochemistry (IHC) staining to examine synaptic density around motoneurons caudal to the lesion site where stimulation or sham stimulation occurred. Synaptic quantification has been completed using FIJI software. Our initial results reveal increased synaptic density around the motoneurons of rats that received optogenetic spinal stimulation, suggesting an increase in synaptic plasticity and connectivity. This indicates that stimulation not only enhances axonal growth but also supports the formation of new connections with downstream neurons partially disconnected by the injury. This study provides insight into the circuitry-related changes involved in SCI recovery. Identifying specific mechanisms of how optogenetic stimulation improves recovery can guide the development of more effective stimulation paradigms and treatment strategies in order to optimize functional recovery for people with spinal cord injury

The use of pesticides in the Pacific Northwest is essential in the process of safeguarding public health, most notably by mitigating pests, protecting our food supply, and aiding in distribution to supermarkets, restaurants, and our homes. However, long-term exposure to pesticides can result in illness for those handling the substances as well as their families. Prior research has shown that current pesticide application methods play a role in accelerating illness. Newer methods, such as aerial drone spraying and “smart” sprayers, involve the use of emerging technologies that are poised to change the landscape of the agricultural industry and health outcomes of farmworkers. Under the supervision of the Pacific Northwest Agricultural Health and Safety (PNASH) Center, my project will be assessing thoughts regarding adoption of these technologies. Through the creation of an electronic REDCap survey, I will be obtaining a variety of responses from agricultural workers, farm decisionmakers, and others involved in the application of pesticides on farms. Once the survey is deployed, I will analyze responses both quantitatively and qualitatively using Dedoose and R statistical methods, respectively. From these responses, I will work with the PNASH team to evaluate the adoption of current and emerging pesticide technologies among Northwest fruit growers, as well as their impacts on occupational health and safety. Through this project, I hope to collect a wide range of perspectives and thoughts regarding the implementation of new pesticide application technologies, particularly unique opinion points (positive and negative) I did not otherwise consider in my initial research with the PNASH Center. The main objective of my research project is to capture the attitudes of the pesticide application technologies to inform policy, regulations, and decision-making regarding their uses.

HIV is characterized by a chronic state of inflammation that may lead to the development of cardiomyopathy, a condition where the heart muscle cannot effectively pump blood. The purpose of this report is to explore the relationship between HIV and the phenotypic expression of hypertrophic cardiomyopathy (HCM). In this patient case study, I analyze the possibility of HIV-related changes in heart structure affecting HCM’s development into heart failure, as well as an increased risk of surgical complications. I examined a 43-year-old female HIV patient who showed symptoms of heart failure and was diagnosed with HCM. She went through various surgical procedures that were complicated by post-pericardiotomy syndrome, an inflammatory response to cardiac surgery. I used the patients' hospital vitals and procedures, as well as current HIV and HCM literature to propose the possible relationship between HCM phenotype and HIV, as well as the role that inflammatory properties play between HIV and the probability of post-surgery complications. HCM phenotype may vary among patients due to its interactions with gender, genetics, and the environment, and can result in heart failure. In this patient, it is possible that there is an increase in severity in HCM phenotype due to the inflammatory factors associated with HIV, further contributing to the development of both heart failure and complications in surgery. The case of this patient illustrates how further research needs to evaluate the specific molecular mechanisms in HIV that could be impacting the expression of HCM, as well as the further development of post-pericardiotomy syndrome.

Diabetic Cardiomyopathy (DCM) is a cardiovascular complication developed from diabetes mellitus, causing a dilation of cardiac ventricle tissue, ultimately impairing systolic function. Although this condition affects 1.1% of the population and nearly 17% of all diagnosed diabetics, mortality rates are exceeding large, with almost 1 in 3 developing death or heart failure after less than a decade. Therefore, effective therapeutics are required to mitigate this prevailing epidemic. Chloride Intracellular Channels (CLICs) are a family of ion channel proteins, permeable to chlorides, which maintain specific cytoplasmic ion concentrations to regulate necessary homeostatic conditions for cellular function. Previous research from our laboratory demonstrates that the absence of CLIC4, a member of the CLIC family and a mitochondrial-associated membrane ion channel in cardiomyocytes, prevents the alteration of sarcomere integrity and beat rate upon high glucose exposure, which is highly prevalent in DCM. Additional research suggests CLIC4 regulates sarcoplasmic reticulum (SR) mitochondrial calcium signaling in cardiomyocytes, and the absence of CLIC4 prevents the occurrence of diabetic cardiomyopathy. However, the mechanism of CLIC4-mediated cardiac dysfunction is still relatively unknown. Previous investigation demonstrates that dysregulation of Ryanodine Receptor 2 (RyR2) function in the SR of cardiomyocytes, due to its oxidation, could augment cardiac dysfunction upon diabetes via modulating calcium levels. This experiment illustrates that pharmacologically blocking CLIC4 reduces oxidative stress in cardiomyocytes, which we measured via staining of cardiomyocytes with DCFDA. Additionally, we found the association of CLIC4 with RyR2 through immuno-pulldown experiments. We anticipate that CLIC4 regulates the oxidative status of RyR2 and modulates its activity in diabetic cardiomyocytes.

Polycystic Ovary Syndrome (PCOS) is the most prevalent reproductive condition in pre-menopausal women, impacting around 5-10% of women in the U.S., despite being underdiagnosed. Previous research has linked a higher free androgen index with cardiovascular risk factors in women with various forms of ovarian dysfunction. Given that elevated androgen levels are a criterion for PCOS diagnosis, understanding the potential association between total testosterone and cardiometabolic risk factors in women with PCOS, specifically, is crucial. This cross-sectional study investigates the relationship between total testosterone levels and cardiometabolic risk factors among women diagnosed with PCOS. Limited research exists on endocrine and cardiometabolic health in PCOS patients, prompting our inquiry. Data for this study was extracted from women with PCOS who attended the University of Washington Endocrinology Clinical and Diabetes Institute. Blood samples underwent analysis for biomarkers including total testosterone, glucose metabolism, and lipid levels. Linear models, both adjusted and unadjusted, were applied to assess correlations between total testosterone levels and the aforementioned biomarkers. Insights into the impact of total testosterone on insulin resistance and lipid levels could offer better insights into how women with PCOS can better manage their health. Preliminary findings indicate a limited correlation between total testosterone and cardiometabolic risk factors, contradicting previous studies. Further analysis, including controlling for factors such as oral contraceptive use, will be done to bring greater clarity to these results. This study aims to bridge gaps in our understanding of the mechanisms by which PCOS can affect the health of women. It also seeks to address the underfunding and underrecognition of research in diseases that primarily affect women. Future research in this domain must be done to investigate biomolecular pathways on how testosterone could potentially affect cardiometabolic health.

Spasticity is an increase in muscle tone (hypertonus) and abnormal muscle stiffness that impedes functional activity. Oftentimes observed among individuals with chronic neurological conditions such as traumatic brain or spinal cord injury (SCI), spasticity develops as a result of damage to the central nervous system (CNS). This damage disrupts the balance of supraspinal inhibitory and excitatory inputs to the spinal cord, which can lead to the loss of inhibitory inputs and hyperexcitation of the spinal reflex arc. The aim of this project is to develop an electrical stimulation protocol that regulates imbalances of supraspinal input and the spinal reflex in order to potentially alleviate spasticity caused by traumatic neural injury in patients. The hyperexcitation associated with spasticity is measured using the Hoffman-reflex (H-reflex). Previous studies have revealed that electrical stimulation of the rat motor cortex can modulate long-term spinal excitability. In this study, behaving noninjured Long Evans rats are implanted with cortical implants to induce stimulation to the motor cortex, grounding electrodes to filter environmental noise, cuff electrodes to evoke the H-reflex, and EMG electrodes to record the H-reflex response. The H-reflex is assessed by stimulating a cuff electrode surrounding the median nerve and measuring the consequent activity of EMG electrodes that are implanted into the flexor, extensor, and tricep muscles before and after electrical stimulation of the motor cortex. Our preliminary results indicate that different frequencies of cortical stimulation can modulate the H-reflex, suggesting that our novel cortical stimulation protocol may reduce spasticity and promote restoration of motor function. In future studies, I plan to assess the efficacy of cortical stimulation for improving spinal excitability in spastic animals following chronic SCI.

Active galactic nuclei (AGNs) are galaxies that consist of a supermassive black hole at the center. Quasars are a type of AGN, which are up to thousands of times more luminous than entire galaxies due to the active accretion of gas onto the supermassive black hole. When active accretion shuts off, a quasar could change into a more normal galaxy. According to standard theory, such significant changes in accretion processes are predicted to occur over timescales of 10,000 to 100,000 years. However, recent studies have discovered a new and unusual type of quasars that undergo dramatic change on timescales of decades to years, contrary to predicted timescales. These objects are dubbed “changing-look quasars” (CLQs). To better understand the physical processes behind CLQs, more CLQ spectra must be identified and examined to perform further statistical analyses. This study analyzes on the order of 102 quasar spectra from the Sloan Digital Sky Survey (SDSS). For each object analyzed, we first assess whether the SDSS spectroscopic pipeline got the classification and redshift correct. In multi-epoch spectra, we then identify spectral features that change over time to distinguish CLQs from regular quasars with relatively constant accretion. This is done by looking at changes in velocity widths of emission lines in at least one of the common quasar emission lines. By producing a larger statistical sample of new CLQs, this study can reveal new information about accretion changes in AGNs, improve our understanding of accretion physics near supermassive black holes, and potentially challenge the standard theory of accretion.

The Manduca sexta hawkmoth, a proficient pollinator, employs its antennae to efficiently navigate its surroundings. With their antennas highly developed olfactory sense as well as their wide range of odor recognition using their sensory receptor cells, moth antennae are an ideal candidate for developing reliable biosensors. In contrast, commonly used artificial sensors are inefficient and inaccurate in chemical detection. Furthermore, their manufacture is challenging due to their inconvenient design for the user. To evaluate the antenna's effectiveness as a biosensor model, we assessed neural activity in the moth antenna by means of an electroantennogram (EAG). To do this, we attached the removed antenna to a circuit to amplify and measure voltage variations across the antennal nerves during odor stimulation. We then placed the circuit into a wind tunnel and administered a selection of odorants over a determined cycle of durations ranging from 0.2, to 10 seconds. The odorants included a floral mixture from Datura flowers and a certain compound in the mixture (linalool) known to be attractive to moths, as well as volatile organic chemicals (VOCs) replicating both healthy and COVID breath. Our findings show strong initial spikes of electrical activity in the receptor cells correlating to odorant release​, favoring the shorter durations and both the floral mixture and linalool. Prolonged exposure (5 and 10 second durations) to odorants caused continuous increased activity in the antennae, with a more pronounced response observed in the COVID VOCs and linalool. These results demonstrate that moth antennas are a suitable model for the construction of highly accurate and efficient biosensors, and support the feasibility of implementing them in devices aimed at detecting and identifying substances of interest. Future work will explore additional COVID-associated compounds and apply data to an algorithm for machine learning software to enhance capabilities for disease diagnosis.

Alzheimer's disease (AD) is the most common type of dementia with an estimated 1 in 85 people affected worldwide. It is characterized by accumulations of amyloid beta which result in difficulties with memory and other cognitive activities. While there are treatments that show promise, these are only effective in the preclinical phase of the disease where there are no symptoms, which take approximately 20 years to appear. Unfortunately, measuring amyloid beta directly is invasive and costly, so functional magnetic resonance imaging (fMRI) is used to detect changes in the brain caused by amyloid beta accumulation. Moreover, established techniques for using fMRI for detecting AD show limited success. Therefore, the goal of my project is to generate biomarkers of the earliest neural communication changes associated with AD to allow the application of treatment when it is most effective. This will be done through the use of a graph diffusion autoregressive (GDAR) model, recently developed in my lab. The GDAR model is capable of estimating dynamic changes of neural communication, but has only been applied to electrical recordings. I have demonstrated its validity for fMRI data using a healthy control group through analyzing the model’s prediction capabilities and test-retest consistency, which are both strong. Furthermore, I correlated the GDAR model’s outputs with the primary method in the field, functional connectivity analysis, showing only loose correlation and the potential for biomarkers that are novel from what is currently known. Currently, I am applying the model to AD patients, and anticipate its outputs to contrast those of healthy patients through the use of unsupervised clustering techniques. This would allow the model’s outputs to be used as biomarkers for AD and improve the feasibility of its treatment and prevention.

In the 20th century, the discovery and widespread use of antibiotics became humanity's primary weapon against pathogenic bacteria. Overuse of antibiotics has unfortunately given rise to antimicrobial resistance, weakening us in this evolutionary arms race. Proteolysis-targeting chimeras (PROTACs) have been proposed as a strategy for development of novel therapeutics. By tagging target proteins with ubiquitin, targeted protein degradation (TPD) can occur via a eukaryote's own molecular machinery. Due to prokaryotes lack of ubiquitin, research has shifted to the development of PROTAC-like molecules to achieve proteolysis and cell death in bacteria, called BacPROTACs. However, to eventually experiment with these small molecules and see their mechanism of TPD, off-target effects, and changes in host and bacterial proteomes, we must be able to profile the degradation of proteins in an unbiased manner. Proteomics and mass spectrometry can identify and measure thousands of proteins simultaneously, enabling systems-level and mechanistic understanding of these novel therapeutics. In order to ensure reliability, reproducibility, and overall accuracy in analyzing a cell’s proteome, an optimized proteomics workflow is imperative. Our lab sought to understand the downstream impacts of different sample preparation protocols, differing in the material used to capture precipitated protein. Here, I present an evaluation of three proteomic sample preparation methods used on triplicates of reduced and alkylated aliquots of human cell lysate: "SP3" (single-pot solid-phase sample preparation), uses magnetic carboxylate beads as a substrate for protein aggregation; "SP4", omits a capture substrate in favor of centrifugation; and "S-Trap/S-Tip," captures protein on a borosilicate glass fibers filters. Following a Trypsin and LysC digest and desalting, samples will be run on an Orbitrap Eclipse mass spectrometer. Analysis and subsequent optimization with more complex human samples will ensure the selection of a protocol providing the highest proteomic coverage for further research.